Compounds That Act To Modulate Insect Growth And Methods And Systems For Identifying Such Compounds

ABSTRACT

Disclosed are methods and systems for screening for compounds that act to modulate insect growth. Bioassays including cell culture and/or transgenic insects engineered with various components of the ecdysoid receptor (EcR) and/or the farsenoid-X receptor (RXR) systems to identify compounds that act as insecticides and/or hormone receptor activators are described. Also described are compounds, and compositions, identified as being putative insecticides based upon their ability to activate EcR and/or FXR mediated transcription.

STATEMENT OF RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/929,090, filed Aug. 27, 2004, entitled “Compounds That ActTo Modulate Insect Growth And Methods And Systems For Identifying SuchCompounds”, which claims priority under 35 U.S.C. §119 from U.S.Provisional Patent Application No. 60/498,847, filed Aug. 29, 2003,entitled “Methods and Systems for Identification of Insecticides andHormone Receptor Activators”; the disclosures of U.S. patent applicationSer. No. 10/929,090, and U.S. Provisional Patent Application 60/498,847are incorporated by reference herein in their entireties.

FEDERAL SUPPORT

The work described herein was supported at least in part by Federalgrants from the U.S. Department of Agriculture Competitive Grantsprogram (00-35302-9327 and 03-35302-13474) and the National Institute ofEnvironmental Health Sciences. Thus, the Federal government may haverights in this invention.

FIELD OF THE INVENTION

The present invention relates to compounds that act to modulate insectgrowth and methods and systems for identifying such compounds.

BACKGROUND

A vast collection of man-made and plant-derived chemicals that functionas insecticides has been amassed over the past sixty years. Concernsover the use of these insecticides, the development of insectresistance, and the possible risk of long-term use for human health havefueled efforts to understand the mechanism by which such compounds act.Some, like the organo-chlorine DTT, inhibit ATP production (Sacklin, J.A. et al., 1955, Science, 122:377-378). Others such as pyrethrins andorganophosphates are neurotoxic (Soderlund, D. M., et al., 2002,Toxicology, 171:3-59; Johnson, M. K., 1975, Arch. Toxicol., 34:259-288).

Insect development appears to be driven by the action of at least twohormone classes, the ecdysteroids and the juvenile hormones (JHs,juvenoids) (Riddiford, L. M., 1994, Adv. Insect Physiol., 24:213-274;Gilbert, L. I., et al., 2000, Insect Biochem. Mol. Biol., 30:617-644;Thummel, C. S., 2002, Insect Biochem. Mol. Biol., 32:113-120). Itappears that ecdysteroids are responsible for initiating metamorphosis,and in some insects, regulating adult fertility. In contrast, JH appearsto be required for reproductive processes such as adult femalevitellogenesis (Wyatt, G. R., and K. G. Davey, 1996, Adv. InsectPhysiol., 26:1-15). Also, the simultaneous presence of ecdysteroids andjuvenile hormone (JH) leads to larval-larval molting.

There are two heterodimeric partners that comprise the functional insectecdysteroid receptor complex: the ecdysone receptor (EcR) (Koelle et al,1991, Cell, 67:59-77) and Ultraspiracle (USP) (Oro, A. E., et al., 1990,Nature, 347:298-301; Henrich, V. C., et al., 1994, Dev. Biol.,165:38-52). Both EcR and USP belong to the nuclear receptor superfamily,which includes receptors for steroid and thyroid hormones, retinoicacid, and fatty acids (Mangelsdorf et al., 1995, Cell, 83:835-839).Also, recently, a second receptor, DHR38, as a heterodimeric partner ofUSP has been shown to mediate ecdysteroid responses, but the mode ofaction is not classical, in that it does not involve direct binding ofthe ecdysteroid to either DHR38 or USP (Baker et al, 2003, Cell113:731-742).

There may also be mammalian counterparts to the insect receptor forectdysteroids. Thus, EcR structurally resembles the vertebrate farnesoidX-activated receptor (FXR) (Forman, B. M., et al., 1995, Cell81:687-695). FXR is a member of the steroid receptor family thatincludes receptors for glucocorticoids, estrogen, vitamins A and D,thyroid hormones, and fatty acids. Also, there is evidence to suggestthat USP may be the insect orthologue of the vertebrate retinoid Xreceptor (RXR) (Oro, A. E., et al., 1990, Nature 347:298-301).Comparisons of amino acids in the FXR and EcR DNA-binding domains reveal60% identity, and the ligand binding domains (LBD) regions of these tworeceptors share about 45% identity. Nevertheless, the possiblefunctional analogy between FXR/RXR and EcR/USP has yet to be resolved.For example, it has been shown that the vertebrate RXR is activated bymethoprene acid but not JHIII or methoprene (Harmon, M. A., et al.,1995, Proc. Natl. Acad. Sci. USA, 92:6157-6160). In contrast, the insectUSP complexes with JHIII and methoprene but not their acid metabolites(Jones, G. et al., 1997, Proc. Natl. Acad. Sci. USA, 94:13499-13503).

Due to concerns about possible toxicity of man-made insecticides, thereis a need to identify natural compounds that have the ability tomodulate insect survival and development. Understanding the basis bywhich compounds interact with the insect EcR and/or FXR to interferewith insect development may provide a rational basis for the isolationof safe, but effective insecticides.

SUMMARY OF THE INVENTION

Embodiments of the present invention provide compounds that act tomodulate insect growth and methods and systems for identifying suchcompounds. The present invention may be embodied in a variety of ways.

In one embodiment, the present invention comprises a method for testingthe ability of a compound to act as a modulator of insect growthcomprising determining whether the compound increases FXR-mediatedtranscription and/or EcR-mediated transcription.

In one embodiment, the method comprises a cell based assay. For example,in one embodiment, the method may comprise transfecting a cell with DNAcomprising a functional isoform of the ecdysone receptor (EcR) or thefarnesoid X-activated receptor (FXR). In one embodiment, a cell thatdoes not include EcR, FXR, or a binding partner for EcR or FXR is usedfor the assay so that there may be minimal background due to endogenousactivation via EcR or FXR. The method may further comprisecotransfecting the cell with a DNA construct comprising a functionalheterologous binding partner for either the EcR or FXR.

Increases in EcR-mediated transcription or FXR-mediated transcriptionmay be measured in a variety of ways. In one example embodiment, DNAcomprising a gene linked to a hormone response element (HRE) thatrecognizes an activated EcR complex or an activated FXR complex may betransfected into the cell. The gene linked to the HRE may be a reportergene that is easily measured, as for example, the chloramphenicolacetyltransferase (CAT) gene. Once the individual components requiredfor EcR-mediated transcription or FXR-mediated transcription are presentin the cell, the compound to be tested for potential ability to increaseFXR-mediated transcription or EcR-mediated transcription may be added,and activation of the gene that is linked to the HRE measured toquantify transcription mediated by either FXR or EcR. In one embodiment,the increase in reporter gene activity may be used to quantify theactivity of the test compound as a potential insecticide, where theability of a compound to act as an insecticide is positively correlatedwith activation of HRE mediated transcription of the reporter gene.

The present invention also comprises a method that may be used to screenfor insecticides in the environment. Thus, in an embodiment, the presentinvention comprises a method for in situ testing for the presence ofcompound having the ability to modulate insect growth comprisingtransfecting a cell in an organism with DNA that encodes a functionalisoform of the farnesoid X-activated receptor (FXR) and/or the ecdysonereceptor (EcR). In one embodiment, the cell may also be transfected witha DNA encoding a functional RXR or USP isoform. Also, the cell maycomprise a DNA comprising a hormone response element (HRE) linked to areporter gene. In one example embodiment, the cell may be exposed to anecdysteroid. The organism may then be exposed to the compound to betested to determine the effect of the compound on FXR-activatedtranscription and/or EcR-activated transcription.

The present invention further comprises assay systems. In oneembodiment, the present invention may comprise an assay system for theidentification of compounds having the ability to act as a modulator ofinsect growth comprising a host cell transfected with an exogenous DNAconstruct comprising sequences encoding a functional isoform of theecdysone receptor (EcR) or the farnesoid receptor (FXR); an exogenousDNA construct comprising sequences encoding a functional heterologousbinding partner for EcR or FXR, wherein the heterologous binding partnerforms a complex with either EcR or FXR, and wherein the complex binds toa hormone responsive element (HRE) on DNA to activate genetranscription; and DNA comprising a reporter construct comprising an EcRor FXR activated hormone response element (HRE) linked to a gene.

The present invention also comprises compounds that act as insecticidesby their ability to inhibit insect growth, reproduction, and/ormorphogenesis. Thus, in another embodiment, the present inventioncomprises a composition for use as an insecticide comprising a compoundthat increases FXR-mediated transcription and/or EcR-mediatedtranscription mixed with a suitable carrier for application to plants.

In yet another embodiment, the present invention may comprise a use forcompounds identified using the methods and systems of the invention asinsecticides and/or modulators of insect growth.

Certain embodiments of the present invention may comprise variousadvantages. For example, the present invention comprises methods andsystems to identify natural compounds derived from plants that have theability to increase FXR-mediated transcription and/or EcR-mediatedtranscription and thus, may be potential insecticides. Such naturalcompounds are potentially non-toxic insecticides; some of the compoundsmay even form part of the human diet.

The present invention may also provide for the discovery and/orrefinement of ecdysteroid agonists and antagonists which exhibit a highpotency for a targeted pest species, while exerting little impact oncolocalized nonpest insects. A baseline of structural and mechanisticinformation about the insect ecdysteroid receptor and potential ligandsalready exists that may serve as a foundation for “rational design”approaches. The diversity of chemical structures associated withecdysteroid agonist and antagonist activity indicates that there may benumerous potential agents having insecticidal or growth modifyingactivities that may be characterized using the systems and methods ofthe present invention. Because many of the compounds of the presentinvention are present in human dietary sources, they may be particularlyuseful for the control of insects that act as vectors or predators ofanimal and human populations. Thus, using the compounds, methods, andsystems of the present invention, it may be possible to developstrategies for refining the effectiveness of existing insecticides aswell as discovering new ones.

Additionally, as plants produce phytoecdysteroids and nonsteroidalagonists/antagonists, the possibility exists for genetic engineering tomaximize and/or enhance the ability of plants to synthesize insecticidesthat impair insect receptor activity; the compounds produced by suchengineered plants may be evaluated using the methods and systems of thepresent invention.

Also, ecdysteroids and their receptor may be implicated in adult insectreproductive development and physiological processes. Thus, thecompounds of the present invention may be potentially useful forcontrolling adult stage as well as developing pests.

Additional features of the present invention will be describedhereinafter. It is to be understood that the invention is not limited inits application to the details set forth in the foregoing or followingdescription but is capable of other embodiments and of being practicedor carried out in various ways.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic diagrams of cell-based assay systems fordetecting insecticides in accordance with example embodiments of thepresent invention where (1A) showns activation of EcR-mediatedtranscription potentiated by an ecdysteroid by compound X, and (1B)shows activation of FXR-mediated transcription by compound X.

FIG. 2 shows the potentiating effects of juvenile hormone III (JHIII)dosage on the muristerone-A (murA) induced response mediated by a GRdEcRchimera and mouse RXR (mRXR) using a chloramphenical acetyltransferase(CAT) reporter construct (EcRE)₅-ΔMTV-CAT in mammalian Chinese HamsterOvary (CHO) cells in accordance with example embodiments of the presentinvention. Sets 1, 2 and 3 correspond to 0, 0.01 and 0.1 μM murA.

FIG. 3 shows the ecdysteroid response and potentiation effects of JHIII(log fold induction based on normalized activity in relative light units(RLUs)) using a luciferase reporter gene (EcRE)₅-ΔMTV-LUC in CHO cells,and measured for various EcR combinations and mouse RXR (mRXR) inaccordance with example embodiments of the present invention, whereinthe constructs used were as follows: (3A) VP16dEcR/mRXR at 0.01 μM murA;(3B) VP16dEcR/mRXR at 0.1 μM murA; (3C) VP16dEcR/VP16CfUSP at 0.1 μMmurA, (3D) EcRA/mRXR at 1 μM murA; (3E) EcRB1/mRXR at 0.1 μM; (3F)EcRB2/mRXR at 0.1 μM murA.

FIG. 4 shows the effects of murA, 20E, and JHIII on RLU activity inducedby (EcRE)₅-ΔMTV-LUC in CHO cells cotransfected with Drosophila EcRisoforms and VP16CfUSP in accordance with example embodiments of thepresent invention where sets 1, 2, and 3 in the figure correspond toEcRA, EcRB1 and EcRB2, respectively.

FIG. 5 shows the response of various combinations of nuclear receptorsand their binding partners to muristerone A and JHIII in CHO cells inaccordance with example embodiments of the present invention, where EcRrefers to VP16dEcR, USP refers to VP16CfUSP, and FXR and RXR refer tonatural mammalian forms.

FIG. 6 shows that FXR may be activated by sesquiterpene metabolites offarnesyl diphosphate in accordance with example embodiments of thepresent invention wherein the relative efficacy of each isoprenoid as aninducer of FXR-dependent transcription is displayed.

FIG. 7 shows molecular structures of natural and synthetic juvenilehormone agonists with FXR effector activities in accordance with exampleembodiments of the present invention including: (7A) farnesol-likeactivators; (7B) juvenile hormone agonists from plants; and (7C)methylenedioxyphenyl-like chemicals with JH activity.

FIG. 8 shows FXR activation profiles for plant secondary metabolites andcongeners in accordance with example embodiments of the presentinvention, wherein the following compounds were tested for FXRactivation: (8A) tea tree oil and constituents α-terpineol, 1,8-cineole,and terpinen-4-ol (added individually at 800 μM or together at 400 μMand compared with increasing amounts of tea tree oil (TTO)); (8B) coffeediterpenes cafestol acetate and kahweol acetate; (8C) cucurbitacin D(cuc D) (1 μM) added to cells with, or without, farnesol (45 μM) orchenodeoxycholic acid (CDCA) (40 μM); (8D) bergamot ingredientsbergamotin, 5-methoxypsoralen, and 8-methoxypsoralen; (8E)methylenedioxyphenyl compounds myristicin, methyleugenol, and safrole;(8F) rotenone and rotenonic acid with a cleaved furan ring; (8G) hopsingredients isoxanthohumol (IX); xanthohumol (XN); and8-prenylnaringenin (8PN); and (8H) xanthines caffeine (CF), theophylline(TP), xanthine (XT), hypoxanthine, and theobromine (TB) (not shown).

FIG. 9 shows FXR-mediated transcription may be increased byphenylpyrazole insecticides in accordance with an example embodiment ofthe present invention, wherein increasing doses of fipronil,chlorfenapyr (pirate), and imidacloprid were tested as indicated for theability to increase FXR-mediated transcription.

FIG. 10 shows that FXR-mediated transcription may be inhibited orstimulated by metabolites or analogues of the JH antagonist precocene inaccordance with example embodiments of the present invention wherein:(10A) shows TLC separation of precocene isoforms and comparison of FXReffector activities; (10B) shows that microsome-treated precocene Igenerates FXR antagonists; (10C) shows molecular structures ofprecocene-like analogs that increase or inhibit FXR-dependent activity;(10D) inhibition of farnesoid (F)-induced FXR-dependent transcriptionmediated by esculetin (E); (10E) shows that FXR-mediated transcriptionis inhibited by ubiquinone-1, wherein the indicated doses ofubiquinone-1 were added to CHO cells transfected with plasmid DNAs tomeasure agonist activity and identical doses were added with 30 μMfarnesol (F) to measure antagonist activity; and (10F) shows thatFXR-mediated transcription is increased by ubiquinone-2, whereinindicated doses of ubiquinone-2 were tested for ability to increaseFXR-mediated transcription and compared with farnesol (45 μM).

FIG. 11 shows that transcriptional activity programmed bymuristerone-primed ecdysone receptors may be potentiated by juvenilehormones and insecticides in accordance with example embodiments of thepresent invention where: (11A) shows potentiation of ecdysone receptoractivity by JH III, where muristerone A (MUR) was added at the indicateddoses in ethanol vehicle, and increasing amounts of JH III (12, 25, or50 μM) were added (underlying triangles), and numbers over bars indicatethe ratio of the GEcEc-dependent activity produced by 50 μM JH III inthe presence of the indicated dose of MUR to the activity produced byMUR alone; (11B) shows that JH III activity may require both EcR andRXR; (11C) shows that the JH agonist juvocimene from basil may be an EcReffector molecule where muristerone A (0.2 μM) was added alone or with10 or 20 μM juvocimene (J) to GEcEc-transfected cells, and farnesol(farn) (45 μM) or juvocimene was added to FXR-transfected cells; (11D)shows that insecticides may potentiate EcR-dependent transcriptionalactivity in the presence (+) or absence (−) of muristerone A, wherecells were incubated with the indicated natural and syntheticinsecticides (added at 25 μM, except endosulfan which was added at 5μM), where the numbers above the bars indicate the ratio of activityfrom cells treated with insecticide plus MUR divided by that treatedwith MUR alone (fold-induction).

FIG. 12 shows that EcR and FXR ligand binding domains may mediate thetranscriptional effects of juvenile hormones and insecticides inaccordance with an embodiment of the present invention where: (12A)shows GGEc chimeric receptor activation in the presence or absence of0.2 μM muristerone A (mur) by farnesol (farn) (25 μM) and its nerolidolisomer; (12B) shows that juvocimene may potentiate muristerone-primedGEcEc and GGEc-dependent activity; (12C) shows that GGEc may mediate thetranscriptional effects of the FXR effector ubiquinone-2 (U2); (12D)shows that GGF chimeric receptor-dependent activation is increased byRXR where CHO cells were transfected with the indicated combinations ofFXR- or RXR-expressing plasmid DNAs and chenodeoxycholic acid (CDCA) wasadded at a final dose of 40 μM (+) or is absent (−); and (12E) showsthat the chimeric plasmid GGF is activated by the natural and syntheticinsecticides cypermethrin, diazinon, dieldrin, precocene, methyljasmonate, and abietic acid added at final doses of 25 μM.

FIG. 13 shows the molecular structures of juvenile hormones isolatedfrom insects, where the chemical formulas for the “naturally-occurring”juvenile hormones JH 0, JH I, JH II, and JH III are compared with thesynthetic juvenoid ZR354, in accordance with an example embodiment ofthe present invention.

DETAILED DESCRIPTION

Embodiments of the present invention provide compounds that function asinsect growth modulators and/or insecticides. The present inventionrecognizes that there are nuclear receptor-gated signaling pathways ininsects and mammals that may uniformly respond to a broad range ofnatural and synthetic insecticides. For example, the compounds of thepresent invention may target the ecdysone receptor (EcR) and/or thefarnesoid X-activated receptor (FXR) to act as insect growth regulatorsand/or to interfere with insect development.

In additional embodiments, the present invention provides and methodsand systems for the identification of compounds that act as insecticidesor modulators of insect growth and/or development. Bioassays assembledusing these receptors may be employed to guide the rational design ofnovel chemicals that specifically target insect pests.

Also described are compositions comprising compounds that function asinsect growth regulators for the control of pests. The compounds used asgrowth regulators may be natural compounds, isolated from everydayplants such as sesame seeds, hops, coffee, and bergamot oil found intea.

The present invention may be embodied in a variety of ways. In oneembodiment, the present invention comprises a method for testing theability of a compound to act as a modulator of insect growth comprisingdetermining whether the compound increases farnesoid X-activatedreceptor (FXR)-mediated transcription and/or ecdysone receptor(EcR)-mediated transcription. In one embodiment, an increase intranscription comprises an increase from a measurable basal level to ahigher level. Alternatively, an increase in transcription may comprisean increase from an undetectable level to a measurable level.

In one embodiment, an increase in EcR mediated transcription comprisespotentiation of hormone activated EcR mediated transcription. Thehormone may comprise an ecdysteroid. For example, in alternateembodiments, the hormone may comprise muristerone A (murA) or20-hydroxyecdysone (20E).

The method may comprise a cell based assay. In one example embodiment,the method may comprise the steps of: (a) transfecting isolated cellswith DNA comprising sequences that encode a functional isoform of theecdysone receptor (EcR) or the farnesoid receptor (FXR); (b)cotransfecting the cell with: (i) DNA comprising sequences that encode afunctional heterologous binding partner for (a), wherein saidheterologous binding partner complexes with either EcR or FXR, andwherein the complex binds to a hormone responsive element (HRE) on DNAto activate gene transcription; and (ii) DNA comprising the reporterconstruct comprising a hormone response element (HRE) linked to a gene;(c) adding the compound to be tested; and (d) measuring an increase inthe synthesis of the reporter gene protein. In one embodiment, an EcRmodulator may be added to the cell such that potentiation of EcRactivity may be measured.

The present invention further comprises assay systems. In oneembodiment, the present invention may comprise an assay system for theidentification of compounds having the ability to act as a modulator ofinsect growth comprising a host cell transfected with an exogenous DNAconstruct comprising sequences encoding a functional isoform of theecdysone receptor (EcR) or the farnesoid receptor (FXR); an exogenousDNA construct comprising sequences encoding a functional heterologousbinding partner for EcR or FXR, wherein the heterologous binding partnerforms a complex with either EcR or FXR, and wherein the complex binds toa hormone responsive element (HRE) on DNA to activate genetranscription; and DNA comprising a reporter construct comprising an EcRor FXR activated hormone response element (HRE) linked to a gene.

In one embodiment, the increase in reporter gene activity may be used toquantify the ability of the test compound to increase EcR-mediatedtranscription or FXR-mediated transcription. In one example embodiment,the ability of the test compound to increase EcR-mediated transcriptionor FXR-mediated transcription may correlated to the potentialinsecticidal activity of the test compound.

In other embodiments of the present invention, transcription of a genethat is downstream of a hormone response element (HRE) that is activatedby FXR and/or EcR may be measured directly. In an embodiment, a reportergene comprising an HRE may be exogenous to the cell and added bytransfection. Alternatively, transcriptional levels of an endogenousgene known to be activated by FXR or EcR may be measured. For example,in one embodiment, transcription may be measured by quantifying mRNAspecific to a gene that comprises a promoter that includes an FXR HRE.Alternatively, transcription may be measured by quantifying enzymeactivity specific to a protein encoded by a gene that comprises apromoter that includes an EcR HRE. Or, the actual amount of proteintranslated from a gene comprising a HRE may be measured by techniquesknown in the art such as sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), or binding of an antibody specific for thetranslated gene product. Other methods to quantify gene transcription(e.g., quantitative PCR amplification of cDNA, RNA hybridization, andthe like) may also be used.

The heterologous binding partner may vary depending upon theDNA-receptor (e.g., FXR or EcR) being used. In one embodiment, theheterologous binding partner may comprise Ultraspiracle (USP).Alternatively, the heterologous binding partner may comprise vertebrateretinoid X receptor (RXR).

The EcR modulator may be one of several compounds known to interact withEcR and its heterologous binding partner. In one embodiment, the EcRmodulator may bind to the ligand binding domain (LBD) of EcR. Or, theEcR modulator may bind to other portions of the EcR polypeptide. Forexample, the EcR modulator may comprise an ecdysteroid. Thus, in anembodiment, the EcR modulator may comprise muristerone A (murA).Alternatively, the EcR modulator may comprise 20-hydroxyecdysone (20E).The modulator may be added to the assay system as a polypeptide. Inadditional and/or alternative embodiments, the modulator may compriseponasterone A, 3-dehydro-20-hydroxyecdysone, ecdysone, makisterone A,nonsteroidal agonist including RH5849, RH59992, and the like. In anotherembodiment, the EcR modulator may be transfected into the cell as a DNAconstruct.

The methods and assay systems of the present invention may be designedto quantify and/or optimize the ability of compounds to interact withvarious isoforms of the nuclear receptor system. For example, in anembodiment, the isoforms of EcR comprise the Drosophila EcRA, EcRB1, orEcRB2 isoforms.

In one embodiment, the methods and assay systems of the presentinvention may employ a cell-based assay that is designed to have minimalbackground due to endogenous ecdysteroids. Thus, in one embodiment, theisolated cells used for transfection may comprise mammalian cells. Or,the isolated cells may comprise insect cells that do not have a EcR orthat contain a non-functional EcR. For example, in one embodiment,insect cells that comprise EcR “knock-outs,” in that the gene for EcRhas been mutated to silence expression, may be used.

Various reporter systems may be used. The reporter construct maycomprise a promoter having multiple hormone response elements (HREs)linked to a gene encoding a detectable gene product. The hormoneresponse elements may recognize activated FXR and/or activated EcR. Theresponse elements that may be used include the heat shock protein 27(hsp 27) EcRE (Riddihough and Pelham, 1987, EMBO J., 6:3729-3734) orother elements such as a palindromic sequence separated by a singlenucleotide (PAL1) or a direct repeat separated by four nucleotides (DR4)or five nucleotides (DR5) (e.g., Vogtli et al., 1998, Nuc. Acids Res.,10: 2407-2414). In a further embodiment, the reporter gene may comprisechloramphenicol acetyltransferase (CAT). Alternatively, the reportergene may comprise luciferase (LUC). Alternatively, the reporter gene maycomprise green fluorescent protein (GFP).

The methods and assay systems of the present invention may comprisechimeric molecular substrates that allow for the analysis of structuraland functional aspects of proteins involved in the modulation of insectgrowth and/or development. By using such chimeric molecular substrates,compounds that are targeted to specific portions of the receptor and/orits binding partner may be developed. Also, in an embodiment, differenthormone response elements may be employed. For example, in oneembodiment, the hormone response element may comprise an EcR responseelement, such as one found in the gene that encodes heat shock protein27 (hsp27). Alternatively, the hormone response element may comprise aFXR response element such as that found in the ileal bile acid bindingprotein gene (Grober, et al., 1999, J. Biol. Chem., 274:29749-29754). Inan embodiment, the response element may be any nucleic acid sequencethat responds to FXR or EcR to stimulate gene transcription.

Also, in an embodiment, site-directed mutagenesis or randomly generatedmutations within the EcR or FXR LBD may be recovered. Such mutations mayinclude sequences that enhance the induced responsiveness of the cellculture system, and/or reduce non-induced transcription levels whilemaintaining or increasing induced transcriptional activity, and/orchange the specificity or induction properties of the receptor for thepurposes of screening a subclass or potential insecticide compounds.

Also, the methods and assay systems of the present invention may use DNAconstructs isolated from various species to develop species-specificinsecticides. For example, it is known that USP or EcR from variousspecies may have different activities. In one example embodiment, theFXR or EcR constructs and/or their respective heterologous bindingpartners may comprise species-specific constructs. Thus, the DNAencoding the ecdysone receptor (EcR) or the farnesoid X-activatedreceptor (FXR) may comprise a chimera of DNA from different insect orvertebrate sources. For example, the chimera may comprise a mammalianactivating domain. Additionally and/or alternatively, the chimera maycomprise a mammalian DNA binding domain (DBD) and/or a mammalian ligandbinding domain (LBD). In alternate example embodiments, the mammalianreceptor domains (e.g., activating, DBD, or LBD) may be derived from amammalian glucocorticoid receptor (GR) or the farnesoid X-activatedreceptor. In another embodiment, the chimera may comprise anEcR-activating domain. Or, the chimera comprises an EcR DNA bindingdomain (DBD) and/or an EcR ligand binding domain (LBD). In one exampleembodiment, the EcR sequences may be derived from an arthropod. In anembodiment, the EcR sequences may be derived from Drosophilamelanogaster. In yet another embodiment, the chimera may comprise aviral protein-16 (VP16) activating domain from pseudorabies virus. Or,the chimera may comprise Chironomus tentans (lower Diptera) (Cf) USPLBD, such as utilized in the VP16CfUSP construct. In other embodiments,portions of the activating domain, DNA-binding domain, and hinge regionmay be deleted from either the natural EcR or USP receptor. Or, one ofthree characterized mammalian (e.g., mouse, rat, or human) RXRs may beused as the heterologous binding partner. Domains (e.g., LBDs) fromother insect species including Manduca sexta (Lepidoptera), Locustamigratoria (Orthoptera), Heliothis virescens (Leptidoptera), Apismellifera (Hymenoptera), Aedes aegypti (lower Diptera), and Tenebriomolitor (Coleoptera) may also be employed.

The present invention also comprises a method that may be used to screenfor insecticides in the environment. Thus, in an embodiment, the presentinvention comprises a method for in situ testing for the presence ofcompound having the ability to modulate insect growth comprising thesteps of:

(a) transfecting a cell in an organism with DNA comprising sequencesthat encode a functional isoform of the farnesoid receptor (FXR) and/orthe ecdysone receptor (EcR);

(b) cotransfecting the cell with: (i) DNA comprising sequences thatencode a functional RXR or USP isoform; and (ii) DNA comprising ahormone response element (HRE) linked to a reporter gene;

(c) optionally, adding an ecdysteroid;

(d) exposing the organism to the compound to be tested; and

(e) measuring an increase in the protein encoded by the reporter gene.

In an embodiment, the organism comprises an insect. For example, in anembodiment, the organism may comprise a transgenic Drosophila engineeredto express heterologous EcR/USP peptides.

A variety of reporter constructs may be used. In an embodiment, thereporter construct may comprise a promoter comprising multiple hormoneresponse elements (HREs) linked to a gene encoding a detectable geneproduct. For example, the reporter gene may comprise luciferase (LUC) orgreen fluorescent protein (GFP).

Also, in an embodiment, different hormone response elements may beemployed. For example, in an embodiment, the hormone response elementcomprises an EcR response element. Alternatively, the hormone responseelement may comprise a FXR response element.

The heterologous binding partner may vary depending upon theDNA-receptor (e.g., FXR or EcR) being used. In one embodiment, theheterologous binding partner may comprise Ultraspiracle (USP).Alternatively, the heterologous binding partner may comprise vertebrateretinoid X receptor (RXR).

The EcR modulator may be one of several compounds known to interact withEcR and its heterologous binding partner. In one embodiment, the EcRmodulator may bind to the ligand binding domain (LBD) of EcR. Or, theEcR modulator may bind to other portions of the EcR polypeptide. Forexample, the EcR modulator may comprise an ecdysteroid. Thus, in anembodiment, the EcR modulator may comprise muristerone A (murA).Alternatively, the EcR modulator may comprise or 20-hydroxyecdysone(20E). In additional and/or alternative embodiments, the modulator maycomprise ponasterone A, 3-dehydro-20-hydroxyecdysone, ecdysone,makisterone A, nonsteroidal agonist including RH5849, RH59992, and thelike. The modulator may be added to the assay system as a polypeptide.In another embodiment, the EcR modulator may be transfected into thecell as a DNA construct.

The assay system may be designed to quantify and/or optimize the abilityof compounds to interact with various isoforms of the nuclear receptorsystem. For example, in an embodiment, the isoforms of EcR comprise theDrosophila EcRA, EcRB1, or EcRB2 isoforms.

Also, as described for the cell-based assay, site-directed mutagenesisor randomly generated mutations within the EcR or FXR (e.g., such as LBDmutants) may be recovered. Such mutations may include sequences thatenhance the induced responsiveness of the cell culture system, and/orreduce non-induced transcription levels while maintaining or increasinginduced transcriptional activity, and/or change the specificity orinduction properties of the receptor for the purposes of screening asubclass or potential insecticide compounds.

Also, the assay may use DNA constructs isolated from various species todevelop species-specific insecticides. For example, it is known that USPor EcR from various species may have different activities. In oneexample embodiment, the FXR or EcR constructs and/or their respectiveheterologous binding partners may comprise species-specific constructs.Thus, the DNA encoding the ecdysone receptor (EcR) or the farnesoidX-activated receptor (FXR) may comprise a chimera of DNA from differentinsect or vertebrate sources. For example, the chimera may comprise amammalian activating domain. Additionally and/or alternatively, thechimera may comprise a mammalian DNA binding domain (DBD) and/or amammalian ligand binding domain (LBD). In alternate example embodiments,the mammalian receptor domains (e.g., activating, DBD, or LBD) may bederived from a mammalian glucocorticoid receptor (GR) or the farnesoidX-activated receptor. In another embodiment, the chimera may comprise anEcR-activating domain. Or, the chimera comprises an EcR DNA bindingdomain (DBD) and/or an EcR ligand binding domain (LBD). In one exampleembodiment, the EcR sequences may be derived from an arthropod. In anembodiment, the EcR sequences may be derived from Drosophilamelanogaster. In yet another embodiment, the chimera may comprise aviral protein-16 (VP16) activating domain from pseudorabies virus. Or,the chimera may comprise Chironomus tentans (lower Diptera) (Cf) USPLBD, such as utilized in the VP16CfUSP construct. In other embodiments,portions of the activating domain, DNA-binding domain, and hinge regionmay be deleted from either the natural EcR or USP receptor. Or, one ofthe three characterized mammalian (e.g., mouse, rat, or human) RXRs maybe used as the heterologous binding partner. Domains (e.g., LBDs) fromother insect species including Manduca Sexta (Lepidoptera), Locustamigratoria (Orthoptera), Heliothis virescens (Leptidoptera), Apismellifera (Hymenoptera), Aedes aegypti (lower Diptera), and Tenebriomolitor (Coleoptera) may also be employed.

The present invention also comprises compounds that act as insecticidesby their ability to inhibit insect growth. Also, the present inventionmay comprise a use for compounds identified using the methods andsystems of the invention as insecticides and/or modulators of insectgrowth.

Thus, in another embodiment, the present invention comprises acomposition for use as an insecticide comprising a compound thatactivates FXR-mediated transcription or EcR-mediated transcription mixedwith a suitable carrier for application to plants.

In an embodiment, an increase of EcR-mediated transcription by thecompound may comprise potentiation of hormone induced EcR transcription.Preferably, the hormone comprises an ecdysteroid. For example, thehormone may comprise murA or 20-hydroxyecdysone (20E).

Various natural compounds may be used as insect growth regulators and/orinsecticides according to embodiments of the present invention. Forexample, in an embodiment, the compound that increases FXR-mediatedtranscription or EcR-mediated transcription may comprise farnesol or afarnesol metabolite. Also, the compound that increases FXR-mediatedtranscription or EcR-mediated transcription may comprise a juvenilehormone mimetic. Also, the compound that increases FXR-mediatedtranscription or EcR-mediated transcription may comprise a plant-derivedJH agonist. In yet another embodiment, the compound that increasesFXR-mediated transcription or EcR-mediated transcription may comprise aninsecticide synergist. The compound that increases FXR-mediatedtranscription or EcR-mediated transcription may also comprise amonoterpene. In another embodiment, the compound that increasesFXR-mediated transcription or EcR-mediated transcription comprises aditerpene. Or, the compound that increases FXR-mediated transcription orEcR-mediated transcription may comprise a triterpene. The compound thatincreases FXR-mediated transcription or EcR-mediated transcription mayalso comprise a furocoumarin. Or the compound that increasesFXR-mediated transcription or EcR-mediated transcription may alsocomprise a phenylpropanoid. In another embodiment, the compound thatincreases FXR-mediated transcription or EcR-mediated transcriptioncomprises a coumarin. Or the compound that increases FXR-mediatedtranscription or EcR-mediated transcription may comprise a flavonoid.Also, the compound that increases FXR-mediated transcription orEcR-mediated transcription may comprises a linoleic acid metabolite.Alternatively, the compound that increases FXR-mediated transcription orEcR-mediated transcription may comprise a polyketide. In yet anotherembodiment, the compound that increases FXR-mediated transcription orEcR-mediated transcription comprises a xanthine.

Also, compounds that increase FXR-mediated transcription or EcR-mediatedtranscription may comprise man-made compounds. For example, the compoundthat increases FXR-mediated transcription or EcR-mediated transcriptionmay comprise an organochlorine.

A. Definitions

The following definitions may be used to understand the descriptionherein. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skin in the art.

The term “a” or “an” as used herein may refer to more than one objectunless the context clearly indicates otherwise. The term “or” is usedinterchangeably with the term “and/or” unless the context clearlyindicates otherwise.

As used herein, a “ligand” is a molecule that interact either directlyor indirectly with a receptor to form a complex.

An “agonist” comprises a compound that binds to a receptor to form acomplex that elicits a pharmacological response specific to the receptorinvolved.

An “antagonist” comprises a compound that binds to an agonist or areceptor to form a complex that does not give rise to a substantialpharmacological response and can inhibit the biological response inducedby an agonist.

“Polypeptide” and “protein” are used interchangeably herein to describeprotein molecules that may comprise either partial or full-lengthproteins.

As used herein, a “polypeptide domain” comprises a region along apolypeptide that comprises an independent unit. Domains may be definedin terms of structure, sequence and/or biological activity. In oneembodiment, a polypeptide domain may comprise a region of a protein thatfolds in a manner that is substantially independent from the rest of theprotein. Domains may be identified using domain databases such as, butnot limited to PFAM, PRODOM, PROSITE, BLOCKS, PRINTS, SBASE, ISRECPROFILES, SAMRT, and PROCLASS.

A “nucleic acid” is a polynucleotide such as deoxyribonucleic acid (DNA)or ribonucleic acid (RNA). The term is used to include single-strandednucleic acids, double-stranded nucleic acids, and RNA and DNA made fromnucleotide or nucleoside analogues.

The term “vector” refers to a nucleic acid molecule that may be used totransport a second nucleic acid molecule into a cell. In one embodiment,the vector allows for replication of DNA sequences inserted into thevector. The vector may comprise a promoter to enhance expression of thenucleic acid molecule in at least some host cells. Vectors may replicateautonomously (extrachromsomal) or may be integrated into a host cellchromosome. In one embodiment, the vector may comprise an expressionvector capable of producing a protein derived from at least part of anucleic acid sequence inserted into the vector.

The term “fusion protein” may refer to a protein or polypeptide that hasan amino acid sequence derived from two or more proteins. The fusionprotein may also include linking regions of amino acids between aminoacids portions derived from separate proteins. Unless specificallystated, there is no required order of linking polypeptides to form afusion protein.

The term “percent identical” or “percent identity” refers to sequenceidentity between two amino acid sequences or between two nucleic acidsequences. Percent identity can be determined by aligning two sequencesand refers to the number of identical residues (i.e., amino acid ornucleotide) at positions shared by the compared sequences. Sequencealignment and comparison may be conducted using the algorithms standardin the art (e.g. Smith and Waterman, Adv. Appl. Math., 1981, 2:482;Needleman and Wunsch, 1970, J. Mol. Biol., 48:443); Pearson and Lipman,1988, Proc. Natl. Acad. Sci. USA, 85:2444) or by computerized versionsof these algorithms (Wisconsin Genetics Software Package Release 7.0,Genetics Computer Group, 575 Science Drive, Madison, Wis.) publiclyavailable as BLAST and FASTA. Also, ENTREZ, available through theNational Institutes of Health, Bethesda Md., may be used for sequencecomparison. In one embodiment, percent identity of two sequences may bedetermined using GCG with a gap weight of 1, such that each amino acidgap is weighted as if it were a single amino acid or nucleotide mismatchbetween the two sequences.

An “effective amount” as used herein means the amount of an agent thatis effective for producing a desired effect. Where the agent is beingused to achieve a insecticidal effect, the actual dose which comprisesthe effective amount may depend upon the route of administration, andthe formulation being used.

As used herein, “modulation of insect growth” includes the modulation ofthe growth of an individual insect or an insect population and includesmodulation of insect reproduction, morphogenesis, and survival.

As used herein, “insect growth” comprises growth and development of anindividual insect and/or an insect population and thus, refers to thegrowth, morphogenesis, and survival of an individual insect, or aninsect population.

As used herein, an “increase in transcription” comprises an increasefrom a measurable basal level to a higher level. Alternatively, an“increase in transcription” may comprise an increase from anundetectable level to a measurable level.

As used herein, “activation of FXR” or “activation of EcR” describesincreasing FXR-mediated transcription or EcR-mediated transcription,respectively.

As used herein, “FXR-mediated transcription” comprises a genetranscription event that requires binding of the activatedfarnesoid-X-receptor to a promoter upstream of the gene beingtranscribed.

As used herein, “EcR-mediated transcription” comprises a genetranscription event that requires binding of the activated ecdysonereceptor to a promoter upstream of the gene being transcribed.

As used herein, “potentiation” of hormone activated transcriptioncomprises an increase in transcription induced by hormones that bind tohormone response elements upstream of the gene being transcribed.

A “hormone response element” comprises a nucleotide region upstream of agene that mediates the effect of a steroid hormone.

An “isoform” is a variant form of a protein that has the same generalfunction as another protein but which may have small differences in itssequence either because it is encoded by a different gene, is expressedby a different promoter in the same gene, or is derived by alternativesplicing of the same pre-mRNA. For example, EcR may exists in at leastthree versions having trans-activating regions that differ in sequenceto provide isoforms EcRA, EcRB, and EcRB2, each of which can activatetranscription of a gene having an EcR HRE in its promoter. EcRA isderived from a different promoter of the EcR gene, and B1 and B2 arederived from alternative splicing of a pre-mRNA.

A “ligand binding domain” is that portion of a protein or polypeptideinvolved in binding of a ligand.

A “juvenile hormone mimetic” is a compound that functions like any oneof the natural juvenile hormones such as JHI, JH II, or JH III. Thenormal physiological functions of the naturally-occurring compounds arecompromised by ectopic or exogenous administration of any number ofjuvenile hormone mimetic compounds.

An “insecticide synergist” is a compound that acts in synergy with aninsecticide to provide a response that is greater than additive.

A “monoterpene” is an acyclic or cyclic C₁₀ hydrocarbon composed of twoisoprene units and their oxygenated derivatives. Common monoterpenesinclude geraniol, limonene, α-pinene, camphor.

A “sesquiterpene” is an acyclic or cyclic C₁₅ hydrocarbon and theiroxidized derivatives composed of three isoprene units.

A “diterpene” is a acyclic or cyclic C₂₀ hydrocarbon and their oxidizedderivatives composed of four isoprene units.

A “triterpene” is a acyclic or cyclic C₃₀ hydrocarbon and their oxidizedderivatives composed of six isoprene units.

As used herein “coumarin” is 2H-1-Benzopyran-2-one. A “furocoumarin” isa psoralen derivative of coumarin such as bergamotin.

As used herein “phenylpropanoid” is a compound derived fromphenylalanine and cinnamic acid.

A “flavonoid” is a phenolic compound build up of two aromatic rings andheld together by a C3 unite. Flavonoids include chalcones, isoflavanoids(rotenone), flavolignans (silybin).

As used herein, a “polyketide” included molecules synthesized formacetyl CoA, propionyl CoA, butyryl CoA, malonyl CoA, methylmalonyl CoA,and ethylmalonyl CaA intermediates.

B. Juvenoids Potentiate Ecdysone Receptor-Dependent Transcription in aMammalian Culture System

The present invention recognizes that a variety of natural and syntheticinsecticides can act to modulate transcriptional activity programmed byFXR. These putative insecticides may also potentiate DrosophilaEcR-mediated transcription in the presence of limiting amounts of anecdysone, thereby providing an assay system for the development of newinsecticides. Thus, the present invention describes compounds thatactivate FXR and/or EcR as potential insecticides. The present inventionalso describes the use of such FXR-activating and EcR-activating and/orpotentiating compounds isolated from plants or plant metabolites asenvironmentally friendly insecticides.

It has been shown that JH may act upon transcriptional activity, and maymodulate the transcriptional regulation of ecdysteroids (Cherbas, L., etal., 1989, Dev. Genet., 10:177-188; Farkas, R., and J. Knopp, 1997,Arch. Insect Biochem. Physiol., 35, 71-83; Hiruma, K., et al., 1999,Dev. Genes Evol., 209, 18-30; Zhou, X., and L. M. Riddiford, 2002,Development, 1290: 2259-2269). Despite its apparent importance forinsect processes, a single receptor for JH has not been definitivelydemonstrated in any insect or developmental period (Wyatt, G. R., and K.G. Davey, 1996, Adv. Insect Physiol., 26, 1-155; Dubrovsky, E., et al.,2002, Insect Biochem. Mol. Biol. 32:1555-1565).

Although a receptor that activates transcription in response toecdysones (EcR) has been reported (Koelle, M. R., et al., 1991, Cell67:59-77), one that mediates the transcriptional effects of JHs stillhas not been defined. The ability of FXR to induce transcription inresponse to farnesol and metabolites such as JH III indicates that thisnuclear receptor homolog of EcR may exhibit pharmacologic featuresexpected of an insect JH receptor (Forman, B. M. et al., Cell 1995,81:687-693).

It appears that both FXR and EcR may interact with a heterologousbinding partner as a prerequisite to activating transcription ofdevelopmental genes. Ultraspiracle (USP), is a nuclear receptor that maydimerize with the ecdysone receptor (EcR) to form the functionalecdysteroid receptor complex (Thomas, H. E., et al., 1993, Nature,362:471-475; Yao, T. P, et al., 1993, Nature, 366:476-479; Riddiford, L.M, et al., 2000, Vitam. Horm., 60:1-73). USP is an insect orthologue ofthe vertebrate retinoid X receptor (RXR) (Oro, A. E., et al., 1990,Nature, 347:298-301), which itself is responsive to the JH agonist,methoprene acid, but not to methoprene itself (Harmon, M. A., et al.,1995, Proc. Natl. Acad. Sci., USA 92:6157-6160). USP has been implicatedas a JH receptor based on its ability to mediate a transcriptionalresponse to methyl epoxyfarnesoate (JHIII) via a direct repeat (DR12)response element in insect cells (Jones, G., et al., 2001, InsectBiochem. Mol. Biol., 32: 33-49; Xu., Y., et al., 2002, Eur. J. Biochem.,269, 6026-6036).

Whereas USP has garnered considerable attention for its possiblemediation of JH effects, little note has been paid to the similarity ofUSP's partner, the ecdysone receptor (EcR), with the vertebratefarnesoid-activated X receptor (FXR). FXR is a nuclear receptor thatstrongly activates transcription (e.g., by about 10-fold to 20-fold) byJHIII when assayed in a mammalian cell culture system (Forman, B. M., etal., 1995, Cell, 81:687-695). FXR induces transcription from a promotercontaining the heat shock protein 27 (hsp27) ecdysone response element(EcRE) (Riddihough, G., and H. R. Pelham, 1987, EMBO J., 6:3729-3734).The ligand-binding domain (LBD) of rat FXR shows about 40% amino acididentity with Drosophila EcR.

The present invention recognizes that there may be overlap between themammalian FXR/RXR system and the insect EcR/USP systems, and providesnumerous plant-derived and man-made JHs that have the ability toincrease transcriptional activity programmed by FXR and/or EcR. Thus, invarious embodiments, the present invention comprises assay systems andmethods to test various EcR isoforms and chimeras as having the abilitymediate a transcriptional response to ecdysteroids and JHs. The abilityto reconstitute ecdysteroid responsive transcriptional effects in anotherwise non-responsive mammalian cell culture (Christopherson, K. S.,et al., 1992, Proc. Natl. Acad. Sci. USA, 89:6314-6318) by transfectionwith EcR may offer a strategy for testing compounds for FXR or EcRinduced transcriptional activity as well as any possible effects ofjuvenoids. Because the assay is performed in mammalian cells whichnormally lack endogenous FXR and EcR activity, components required foractivity may be controlled.

Thus, the present invention comprises methods and systems to evaluatethe ability of specific compounds to activate either FXR or EcR mediatedtranscription, wherein compounds exhibiting the ability to activate EcRor FXR mediated transcription comprise potential modulators of insectgrowth and/or insecticides. Example embodiments of assay systems of thepresent invention are depicted schematically in FIGS. 1A and 1B. Theassay systems as depicted in FIGS. 1A and 1B are non-limiting in thatthe mechanism of interaction of the various assay components may varyfrom that depicted in the drawing, while still providing a functionalassay system. For example, in one embodiment, the compound to be tested,(X), may bind directly to FXR or EcR protein as indicated schematicallyin FIG. 1. Alternatively, compound (X) may modify FXR (or EcR) activitywithout directly binding to the protein, but indirectly in some manner.

As shown in FIG. 1A, the assay system may comprise four components. Thefirst component may comprise a DNA construct that encodes a functionalEcR. For example, in one embodiment, a cell (shown as the large oval inFIG. 1) may be transfected with a plasmid comprising sequences thatencode an EcR polypeptide. In one embodiment, the EcR polypeptide maycomprise an EcR chimera comprising a mammalian (e.g., human)glucocorticoid receptor (GR or hGR) trans-activation domain (A/B)attached to an insect (e.g., Drosophila) EcR DNA binding domain andhinge (DBD) (C/D) and ligand binding domain (LBD) (E). Also, in somecases the construct may contain a region (F) that may function as atransactivation domain (Palli, S. R. et al., 2003, Eur. J. Biochem.,270:1308-1315) The second component of the assay may comprise thebinding partner needed for EcR (or FXR) activity. In one embodiment, acell may therefore be co-transfected with a second expression plasmidcomprising sequences that encode a mammalian RXR (e.g., mouse, rat, orhuman RXRα) or an insect USP. These proteins may also comprise atrans-activating domain (A/B) and a DNA binding domain (C) and hinge(D), and a ligand binding domain (E). In an embodiment, RXR and/or USPmay not necessarily comprise an (F) domain. The plasmids may beco-transfected into a mammalian cell, such as a Chinese Hamster Ovary(CHO) cell, so that there is no endogenous EcR or ecdysteroid present.The third component of the assay system may be an exogenous ecdysteroidsuch as muristerone A (murA), that may act to induce EcR dependenttranscription. Finally, the assay system may comprise a means to measureEcR- or FXR-mediated transcription. The response of transfected cells toa compound (X) that is able to increase FXR-mediated transcription orEcR-mediated transcription may be measured using a reporter plasmidbearing a EcR (or FXR) hormone responsive element (HRE). In oneembodiment, the reporter plasmid may comprise a construct havingmultiple copies of the hormone-responsive element inserted upstream of agene having a measurable gene product. In one example embodiment,multiple copies of an ecdysone-responsive element, hsp27 EcRE, areinserted into the mouse mammary tumor virus (MTV) promoter positionedupstream of the bacterial chloramphenical acetyltransferase gene togenerate an (EcRE)₅-ΔMTV-CAT reporter plasmid. The compound to be tested(X) may then added to the cells, and the effect on FXR or EcR activatedtranscription of the CAT gene is determined.

In an alternative embodiment, the reporter gene may comprise theluciferase gene (LUC) or a green fluorescent protein (GFP). For example,in an embodiment, the reporter plasmid comprises an (EcRE)₅-ΔMTV-LUCconstruct produced as described herein. Using the luciferase gene mayallow for FXR or EcR activated transcription to be measured in-situ bymonitoring the luminescence of the cells or organism being used in theassay.

FIG. 1B shows a non-limiting example embodiment of an assay system forFXR-mediated transcription and measurement of the ability of a compound,(X), to modulate FXR-mediated transcription. In this assay system, ahormone, such as murA, may not be required. Thus, in the assay systemshown in FIG. 1B, compound (X) may modify transcription of the reportergene that is mediated by FXR, but that does not require or employ murA.As described for FIG. 1A, the compound to be tested, (X), may binddirectly to FXR or EcR protein, or it may modify FXR (or EcR) activitywithout directly binding to the protein, but indirectly in some manner.

For example, cells may be transfected with plasmids comprising a GRdEcRchimera that consists of the rat glucocorticoid receptor (GR) activationdomain (A/B) attached to the Drosophila melanogaster (d or Dm) EcR DNAbinding domain (DBD) (C/D) and ligand binding domain (LBD) (E), and asecond plasmid comprising mouse RXR (mRXRα). The response of transfectedcells to the ecdysteroid, murA, may be measured using a (EcRE)₅-ΔMTV-CATreporter plasmid that carries five tandem repeats of the hsp27 EcRElinked to the MTV promoter and the chloramphenicol acetyltransferase(CAT) gene. For example, using the assay system of the presentinvention, a response (an increase in CAT activity) is detected uponaddition of the EcR ligand, muristerone A (murA), at doses as low as 0.1μM (FIG. 2). For FIG. 2, sets 1, 2 and 3 correspond to 0 mur A, 0.01 μMmurA and 0.1 μM murA. In an embodiment, only amounts of hormone (e.g.,muristerone A) that comprise submaximal levels of induction of EcRtranscription are added to the assay system to detect signals from testcompounds. Thus, in alternate embodiments, the dose of hormone may rangefrom 0.001 μM to 5.0 μM, or from 0.005 μM to 0.5 μM, or from 0.01 μM to0.1 μM.

In an embodiment, the transfected cells comprise mammalian cells. Byusing mammalian cells as the host, cofactors and transcription factorsare present, but there may be minimal background activity due toendogenous ecdysone receptor and/or ligands. Any cell type that does notnormally respond to ecdysteroids, but having the required transcriptionfactors is a potentially suitable cell type for the assay.

Various combinations of the steroid activation pathway may be employedin the assay of the present invention. Thus, in one embodiment, thepresent invention comprises the use of specific receptor isotypes toevaluate potential insecticides. For example, in an embodiment, theassay may use an arthropod (e.g., insect) ecdysone receptor (EcR), aninsect USP, and a suitable reporter gene construct (e.g., CAT orluciferase linked to a HRE specific to FXR or EcR). Alternatively, theassay may comprise using an insect EcR with a mammalian RXR, and asuitable reporter gene construct. In yet another embodiment, the assaymay comprise use of an FXR construct in combination with a mammalian RXRconstruct. Or, the assay may comprise using FXR in combination with USP.Several non-limiting example embodiments for assay systems of thepresent invention are summarized in Table 1.

TABLE 1 Ligand Assay binding Heterologue Reporter construct HormoneSystem Application GCdEcR USP (EcRE)₅-ΔMTV- Yes Mammalian Cell-basedassay for LUC/CAT insecticides VP16-EcR USP (EcRE)₅-ΔMTV- Yes MammalianCell-based assay for LUC/CAT insecticides GCdEcR RxR (EcRE)₅-ΔMTV- YesMammalian Cell-based assay for LUC/CAT insecticides VP16EcR RxR(EcRE)₅-ΔMTV- Yes Mammalian Cell-based assay for LUC/CAT insecticidesFXR RxR (EcRE)₅-ΔMTV- No Mammalian Environmental Screening LUC FXR RxR(EcRE)₅-ΔMTV- No Mammalian Cell based assay for CAT/LUC insecticide EcRAUSP (EcRE)₅-ΔMTV- Yes Mammalians Cell-based assay for LUC/CATinsecticides EcRB1 USP (EcRE)₅-ΔMTV- Yes Mammalians Cell-based assay forLUC/CAT insecticides EcRB2 USP (EcRE)₅-ΔMTV- Yes Mammalians Cell-basedassay for LUC/CAT insecticides

As indicated in Table 1, the DNA constructs used for the presentinvention may be chimeras. Nuclear receptors, such as EcR and FXR, maycomprise three general domains structural domains: (1) a transcriptionalactivating domain—A/B; (2) a DNA binding (DBD)—C; and (3) a hinge (D)and ligand binding domain (LBD) (E) (FIG. 1). As used herein, the hinge(D) may be considered as part of the DBD (C) or the LBD (E) whendescribing various nuclear receptor protein constructs. Also, for somereceptors, a C-terminal (F) domain may comprise trans-activationcharacteristics. For example, in one example embodiment, the EcRconstruct, GCdEcR, may comprise the human glucocorticoid receptor (GC)or (hGC) activating domain (e.g., A/B) and the Drosophila (d or Dm) EcRDNA binding domain (C) and the Drosophila hinge-ligand binding domain(D/E). Or, a VP16EcR construct comprising the VP16 activating domain(VP16) in combination with Drosophila EcR DBD and LBD domains (EcR) maybe used. The assay may also comprise a construct, GGEc, comprising thehuman glucocorticoid receptor (G) trans-activating domain and DBD withthe Drosophila EcR (Ec) LBD. The assay may also comprise a construct,GEcEc, comprising the human glucocorticoid receptor (G) activatingdomain and the Drosophila (Ec) DBD and LBD. In yet another embodiment,the assay may comprise a construct, GGF, comprising the humanglucocorticoid receptor (G) activating domain and DBD with the FXR (F)LBD derived from vertebrate species (avian, rodent, human). In yetanother embodiment, the assay comprises a construct, VP16CfUSP,comprising the VP16 activation domain directly linked to the LBD (i.e.,D/E domains) of Choristoneura fumerifana (Cf) (spruce budworm) USP. Inanother embodiment, the VP16 trans-activating domain may be linked tothe Drosophila USP protein DBD and LBD to generate the construct,VP16-DmUSPDBD-DmUSPLBD. Or, natural isoforms of Dm EcR with A/B domainsA, B1 or B2 with the F domain deleted (Mouillet et al., 2001) may beused. Table 2 provides several non-limiting constructs that may be usedwith the methods and systems of the present invention, utilizingnomenclature that is standard in the art.

TABLE 2 Receptor Domain Composition of Plasmid Expression Vectors D/E -Hinge- C - DNA Ligand Binding Binding Domain Domain Construct A/B -Trans-Activation (DBD) (LBD) GCdEcR or GEcEc or hGR Dm EcR Dm EcRGRdEcR* GGEc hGR hGR Dm EcR GGF hGR hGR FXR VP16EcR VP16 Dm EcR Dm EcRVP16CfUSP VP16 — Cf USP VP16DmUSPDmUSP VP16 Dm USP Dm USP EcRA EcRA DmEcR Dm EcR EcRB1 EcRB1 Dm EcR Dm EcR EcRB2 EcRB2 Dm EcR Dm EcR VP16USPF1VP16/DmA/B fusion Dm USP Dm USP VP16USPF2 VP16 Dm USP Dm USP VP16 USPF3VP16 — Dm USP mRXRα Mouse RXR Mouse RXR Mouse RXR *As used herein,glucocorticoid receptor elements may be designated as G, GC, or GR.

Also, standard RXR and USP expression plasmids may be used in the assayof the present invention. As is known in the art, expression plasmidsmay have promoter elements and polyadenylation signals between whichnucleic acid sequences encoding the amino acids of interest arepositioned. For RXR and USP expression plasmids, the plasmids produceRXR or USP proteins in the presence of the appropriate transcriptionfactors. Promoter elements for the RXR and USP proteins may be such asto drive transcription in a constitutive fashion. These typicallyinclude those from Rous sarcoma virus or cytomegalovirus.

The use of chimeric DNA constructs allows for the construction ofnuclear receptors which interact with mammalian transcriptional factorsto induce or repress transcription mediated by the DBD and/or LBD of theinsect receptor, EcR, and its partner, USP. Because the mammalian cellsdo not contain endogenous EcR or USP, there is little or no endogenousresponse to the experimental ligand.

The A/B domain of EcR may be responsible for modifying transcriptionalactivity of the receptor. In one embodiment, various isoforms of the EcRA/B domain (e.g., A, B1, or B2) may be used for the EcR construct(Tables 1 and 2 and described herein). In one embodiment, the system maydisplay specificity between a particular EcR isoform and theheterodimeric partner or the ability of the EcR to respond to the testagent (X). Conversely, different EcR/USP and EcR/RXR heterodimericcombinations display different responsiveness to the test agent (X).

In an embodiment, juvenile hormones (JHs) can potentiate the effect ofEcR ligands. For example, a GRdEcR chimera, that consists of the humanglucocorticoid receptor activation domain (GR) attached to theDrosophila melanogaster (d) EcR DBD and LBD was cotransfected with mouseRXR (mRXR) into CHO cells. Using an (EcRE)₅-ΔMTV-CAT reporter gene, itwas found that juvenile hormone III (JHIII) may potentiate the responseof murA in a dose-dependent manner (using 20, 40, 80, and 160 μM JHIII)at submaximal ecdysteroid dosages (0.1 μM and 1 μM murA) (FIG. 2, sets 2and 3, respectively). Also, although JHs can potentiate the effects ofecdysteroid ligands, JHIII may not increase the response above themaximal response seen with high concentrations of the ecdysteroid. Thus,using the assay described for FIG. 2, JHIII does not evoke a responsethat is greater than the maximal level induced by 10 μM murA.

The use of a mammalian cell-based assay system allows for controlledaddition of the components required for activity. Thus, in oneembodiment, juvenile hormones do not interact with unbound EcR, butrequire addition of submaximal levels of exogenous hormone for activity.For example, despite the structural resemblance between the LBDs of EcRand the vertebrate FXR, which is highly responsive to JHIII alone(Forman et al., 1995, Cell, 81:687-693), JHIII alone shows no effect ontranscription mediated by the GRdEcR chimera with RXR (FIG. 2, set 1).

Using the assay system of the present invention, the interaction betweenvarious molecular components of the insect developmental pathway may beoptimized. For example, ecdysteroid responsiveness and JHIIIpotentiation in EcR chimeras may depend, at least in part, upon theactivating ligand being used. Ecdysteroid responsiveness and JHIIIpotentiation in EcR chimeras may also depend upon and the heterodimericpartner used in the assay system. For example, transfection of mammalianCHO cells with the VP16 activation domain linked to the DBD and LBD ofDrosophila melanogaster (Dm or d) EcR, may result in a sensitive androbust ecdysteroid response to muristerone A.

FIG. 3 shows the ecdysteroid response and potentiation effects of JHIIImeasured for various EcR combinations and mouse RXR in accordance withan embodiment of the present invention. As shown in FIG. 3, mouse RXRwas used as the heterologous dimer and a luciferase construct,(EcRE)₅-ΔMTV-LUC, was used as the reporter gene. Shown in FIG. 3 are:VP16dEcR/mRXR at 0.01 μM murA (3 a); VP16dEcR/mRXR at 0.1 uM murA (3 b);VP16dEcR/VP16CfUSP at 0.1 μM murA (3 c); EcRA/mRXR at 1 μM murA (3 d);EcRB1/mRXR at 0.1 μM (3 e); and EcRB2/mRXR at 0.1 μM murA (30, each inthe presence, or absence, of 80 μM JHIII.

It may be seen that the various constructs may display differingactivity profiles. For example, VP16dEcR (viral transactivation A/Bdomain linked to Drosophila (EcR) LBD and DBD) partnered with mouse RXRmay respond to 0.01 μM and 0.1 μM murA (FIGS. 3 a and 3 b). The responseof the VP16dEcR/mRXR system is generally strong and robust as comparedto other combinations of binding and transcriptional activation units.Also, the response of the VP16dEcR/mRXR system may be furtherpotentiated by JHIII. The VP16dEcR chimera may also display adiscernible response to 20E at 10 μM (e.g., over 20-fold) using RXR.VP16dEcR/20E activity is not, however, necessarily potentiated by JHIII.In one embodiment, VP16dEcR/RXR in the presence of 20E is only minimallyaffected by the additional presence of JHIII (not shown).

As described herein, the assay of the present invention may allow forthe mixing of different molecular constructs as a means to betterunderstand the molecular nature of insect development and insecticidestructure and function. In one embodiment, the VP16dEcR construct mayalso be tested with VP16CfUSP (Table 2) (FIG. 3 c). Using this system,the same degree of potentiation may occur for murA using the VP16EcR/USPcombination as with the VP16dEcR/RXR complex, except that a higher murAdose may be required to achieve the same efficacy. Although thenormalized level of JHIII-mediated potentiation of the murA response issimilar with either RXR or VP16CfUSP, the combination of VP16dEcR withVP16CfUSP may shows little to no response to 10 μM 20E and is notaffected by the additional presence of JHIII (not shown).

The assay of the present invention provides for the use of differentecdysone receptor isoforms, either natural or generated by mutagenesis,as a means to evaluate the activity of various test compounds. In analternate embodiment, the assay may comprise the use of various FXRisoforms, either natural or generated by mutagenesis (Zhang, Y, et al.,2003, J. Biol. Chem. 278: 104-110). To evaluate the activity of thevarious EcR isoforms, selected EcR isoforms, such as the three naturalDrosophila melanogaster EcR isoforms, EcRA, EcRB1, and EcRB2, may becotransfected into CHO cells with either USP or RXR expression plasmidssuch as, but not limited to, the VP16CfUSP and/or VP16CRXR fusionproteins. The different EcR isoforms (e.g., Drosophila A, B1, and B2)may be selected to differ at the N-terminal region of the protein, whichis the part of the protein involved in dimerization of EcR with eitherUSP, RXR or other appropriate partners. In an embodiment of the assay ofthe present invention, the isoforms (e.g., A, B1, and B2) may displayunique expression profiles among each other and as compared toVP16dEcR/RXR (not shown).

The activity observed among the three EcR isoforms may depend, at leastin part, upon the identity of the heterologous partner used. Forexample, when tested in the absence of hormone, the EcRB1/VP16CfUSPcombination shows a relatively high level of ligand-independenttranscription, with between 10 and 20-fold higher basal levels thanother EcR constructs. Also, the EcRA isoform in combination withVP16CfUSP may also show a basal level of transcription that is 2 to 3fold higher than the basal level of transcription obtained with EcRB2isoform. In contrast, the basal activity of the EcRB2/VP16CfUSP dimermay be about the same as the basal activities produced by VP16dEcR/RXRand GREcR/RXR (not shown).

In an embodiment, with VP16CfUSP, all three Drosophila isoforms (A, B1and B2) may be induced by about 30-40 fold at 1 μM murA. Also in anembodiment, the response of all EcR isoforms in the presence ofVP16CfUSP is potentiated by the presence of 80 μM JHIII in the presenceof 0.1 μM murA. Data showing induction of isoforms A (set 1), B1 (set 2)and B2 (set 3) by murA, and potentiation by JHIII is shown in FIG. 4. Ina further embodiment, the JH potentiation is dose-dependent, similar tothe results seen with GrdEcR.

Responsiveness of the natural EcR isoforms to the ecdysteroid 20E mayrequire USP (rather than RXR) as a dimeric partner. In yet a furtherembodiment, among the three isoforms (EcRA, EcRB1, and EcRB2), JHIIIpotentiation in the presence of 20E may occur only with EcRB2 isoformand USP. For example, at a dosage of 10 μM 20E, all three Drosophila EcRisoforms and VP16CfUSP generate a consistent and discernibletranscriptional response. Only the EcRB2/VP16CfUSP dimer (FIG. 4, set 3)is potentiated significantly by the additional presence of JHIII,however. That only the B2 EcR isoform is potentiated by JHIII in thepresence of 20E indicates that activation by JHIII may depend upon boththe N-terminal domain of EcR and the activating ecdysteroid.

Specific combinations of the EcR N-terminal domain and the heterodimericpartner (e.g. VP16 and RXR, B2 and USP) may therefore result in afunctional receptor that is capable of showing an ecdysteroid responseand/or JHIII potentiation. Also, in one embodiment of the assay system,the potentiation by JHs is not due to the activation of RXR by eitherJHIII or a JHIII metabolite, as JHIII by itself shows no potentiation oftranscriptional activity.

The differential ecdysteroid- and JHIII-dependent transcriptionalactivities noted among the EcR isoforms may offer insights concerningthe lack of correspondence between cellular isoform titers anddevelopmental effects in Drosophila tissues. The β isoforms may befunctionally distinguished from the EcRA isoform (Bender, M., et al.,Genetics 91:777-788), and recent studies have distinguished biologicalroles for B1 and B2 (Cherbas, L., et al., 2003, Development130:271-284). Also, the B2 N-terminal domain is shorter than the B1N-terminal domain, and is capped with an amphipathic helix (Talbot, W.S., et al., 1993, Cell 73: 1323-1337; Hu et al, 2003). Interestingly,alternative isoforms in the rat FXR also differ greatly in their abilityto mediate ligand-dependent transcriptional activity (Zhang, Y, et al.,2003, J. Biol. Chem. 278: 104-110).

In selected embodiments of the present invention, combinations ofEcR/USP, EcR/RXR, and FXR/RXR show a response to murA that may bepotentiated by JH (FIG. 5). In contrast, the combination of FXR with USPmay not respond to JHIII. Thus, whereas the FXR activator JHIII canpotentiate the transcriptional response of EcR induced by ecdysteroids,it appears that in at least some embodiments, the JHIII responseexhibited by FXR may require RXR, and not USP, as a heterodimericpartner. Thus, in at least some systems, USP and RXR may not beinterchangeable. Also, the presence of a ligand-bound EcR may be aprerequisite for observing the potentiative effects of JHIII on EcR.Assays using the various EcR forms indicate that multiple factors mayinfluence receptor activity, including the activating ecdysteroid, theN-terminal domain of EcR, and the heterodimeric partner.

In one embodiment, the combination of FXR and USP may evoke a low levelresponse in CHO cells to JHIII (FIG. 5) attributable to endogenousexpression of low levels of RXR in these cells, where the addition ofecdysteroids (murA or 20E) with JHIII induces no further elevation ofFXR/USP-mediated activity (FIG. 5). Also, EcR and USP may be unable topotentiate a response to 20 μM chenodeoxycholic acid (CDCA), the mostefficacious naturally-occurring activator of FXR known to date.

A summary of ecdysteroid responsiveness and JHIII potentiation amongvarious combinations of EcR with either RXR or USP proteins (Table 3)shows that murA may exhibit activity with a wider range of ecdysteroidreceptor heterodimers and may be more potent than 20E. Further, the EcRcombination may influence responsiveness. For example, all threeisoforms (EcRA, EcRB1, and EcRB2) may be potentiated by JHIII when murAis the activating ligand. In contrast, for 20E, only the B2/USP may besubstantially potentiated by JHIII. Also, in an embodiment, of the threeisoforms, only EcRB1 acts with RXR. In contrast to the activity seenwith murA, the three isoforms may respond to 20E only in conjunctionwith their natural partner, USP. Thus, it appears that 20Eresponsiveness may involve a compatibility between the N-terminal domainof EcR and the heterodimeric partner. In one embodiment, the high levelof B1 basal activity may relate to a cell-specific aspect of CHOcultures, as this effect has not been seen in HeLa cells. Theselectivity of the 20E responsiveness may be further substantiated bythe ability of the VP16dEcR/VP16CfUSP combination to respond only tomurA, but not to 20E.

TABLE 3 A summary of ecdysteroid responsiveness and JHIII potentiationamong various combinations of EcR with either RXR or USP proteinsEcdysone Receptor MurA + 20E + MurA JHIII 20E JHIII Description RXR USPRXR USP RXR USP RXR USP GRdEcR + n.d + n.d − n.d. − n.d. VP16dEcR++ + + + + − − − EcRA − + − + − + − − EcRB1 + + + + − + − − EcRB2 − +− + − + − + Proteins are described in Examples 1 and 2. (+) designates achange in transcriptional level that exceeds 2.5-fold, (++) indicates aresponse at a lower dosage than other EcR forms. Responses are based ondosages of 0.1 μM murA, 10 μM 20E, and 80 μM JHIII. For murA and 20E,(+) indicates inducibility; for columns involving JHIII, (+) indicatesobserved potentiation that exceeds 2-fold above the levels observed withecdysteroid alone.

The activating ecdysteroid may also determine the ability of JHIII topotentiate a response in other EcR dimers. For instance, theVP16dEcR/RXR combination is responsive to both 20E and murA; but when20E is used, JHIII may not be capable of potentiating the response,whereas JHIII strongly potentiates the murA response.

In an embodiment, EcR, bound to its cognate ligand, may acquire aconformation that allows further activation by JHIII either directly orvia an indirect interaction. Also, the amount of ligand-bound EcR may belimiting factor in the cell-based assay of the present invention. Forexample, while the amount of potentiation JHIII potentiation may remainconstant over a range of submaximal murA doses, the absolutetranscriptional activity attributable to a fixed JHIII dose may increaseas ecdysteroid molarity increases, indicating that the number ofligand-activated EcR proteins may be a rate-limiting factor.

The mechanism of JHIII potentiation for EcR/USP may be different fromeffects of JH analogues on RXR. RXR may be activated through its LBD bymethoprene acid, a metabolite of methoprene, and with the retinoic acidreceptor (RAR) generates a response to the ligand through a directrepeat element (Harmon et al., 1995). Known RXR ligands may alsoincrease the responsiveness of VP16dEcR to murA via the hsp27 responseelement (Saez, E., et al., 2000, Proc. Natl. Acad. Sci. USA,97:14512-14517) to supra-maximal levels. In contrast, potentiation ofEcR by JHIII occurs at a submaximal hormone response through alreadyactivated EcR molecules. Also, RXR ligands activate the VP16EcR/RXRcomplex even when ecdysteroids are not bound to the EcR partner (Saez etal., 2000). By contrast, the effects of JHIII in the assay of thepresent invention may require the simultaneous presence of ecdysteroidsfor any response to be observed.

B. The Farnesoid X-Activated Receptor (FXR) and Ecdysone Receptor (EcR)as Strategic Targets for the Development of Compounds that ModulateInsect Growth

Farnesol was the first molecule to be recognized as a juvenoid (PSchmialek, 1961, Z. Naturf, 16b:461-464). For example, the most widelydistributed juvenile hormone (JH) in the arthropod world, JH III, wassynthesized via bioassay-guided structural optimization based uponfarnesol (Bowers, W. S., et al., 1965, Life Sci., 4:2323-2331) prior toits identification from insect extracts (Roller, H., et al., 1967,Angew. Chem. Int. Ed., 6:179-180). The use of rational design hasprovided a platform for the development of numerous drugs and vaccinesimportant for human and veterinary health, and provides a conceptualbasis for the future design of more effective insecticides. In rationaldesign, biologically important molecules are characterized by comparingstructure with activity in order to identify functional groups thateither enhance, alter, or block activity. Farnesol is metabolized fromfarnesyl diphosphate, a precursor to cholesterol, ubiquinones,dolichols, and other growth-requiring isoprenoids (Goldstein, J. L., andM. S. Brown, 1990, Nature 1990, 343:425-430). Using the methods andassay systems of the present invention, it has been determined thatcompounds derived from everyday plants, such as farnesol and otherjuvenoids, and including those that may constitute a portion of thehuman diet, may comprise the ability to activate FXR and/or EcR (i.e.,to increase FXR-mediated transcription or EcR-mediated transcription,respectively) and thus, may function as insecticides and/or insectgrowth regulators.

In one embodiment, the compounds that increase FXR and/or increase orpotentiate EcR-mediated transcription comprise farnesol metabolites. Asshown in FIG. 6, farnesyl diphosphate is metabolized to juvenile hormoneIII (FIG. 6), and at least some of the intermediate metabolites mayinteract with FXR to induce RXR mediated transcription. For example,farnesol, nerolidol, and JHIII may induce FXR greater than 10-fold.Also, in an embodiment, farnesol, farnesoic acid, and methyl farnesoate,also interact with FXR to induce RXR mediated transcription.

Also, the compounds that increase FXR-mediated transcription, and/orincrease or potentiate EcR-mediated transcription, may comprise juvenilehormone mimetics. For example, FXR-activating farnesoids have beendescribed as JH agonists in insect bioassays (Schneiderman, H. A., andL. I. Gilbert, 1964, Science, 1964, 143:325-333). Thus, in one exampleembodiment, the JH mimetics comprise farnesol, nerolidol, and phytol(Table 4), as well as the synthetic juvenoids methoprene, pyriproxyfen,and the ethyl ester of 7,11-dichloro-2-ene farnesoic acid (FIG. 7A).

TABLE 4 JH and FXR Activities of Isoprenoids and Chemicals JH ACTIVITYaFXR ACTIVITY ISOPRENOID (units/g) (fold-induction) cecropia oil 1000N.T. phytol 32 3 isophytol 0 N.T. all-trans farnesol 140 9 farnesal 32 2farnesyl acetate 5.4 12 farnesenic [farnesoic] acid 7.8 3hexahydrofarnesol 0 N.T. nerolidol 8.9 9 linalool 0.08 1 geraniol 0 1geranyl linalool 0.14 N.T. solanesol 0.05 N.T. juvenile hormone III — 20methoprene — 15 pyriproxyfen — 9 fenvalerate — 12 ^(a) From Schneidermanand Gilbert, Science 143: 325-329 (1964). N.T. = not tested; FXRactivity of “1” indicates that the FXR-dependent transcriptionalinduction was less than 2-fold when tested at doses below cytotoxicity.Chemicals were tested at a final dose of 50 ∝M.

In another embodiment, the compounds that increase FXR-mediatedtranscription, and/or increase or potentiate EcR-mediated transcription,comprise plant-derived JH agonists. In one example embodiment, the plantderived JH agonists that increase FXR-dependent transcription, and/orincrease or potentiate EcR-mediated transcription, comprise echinaceaoil, echinolone, juvocimene, juvabione, α-bisabolol, olive oil,2-hydroxyphenethyl alcohol, 3-hydroxyphenethlyl alcohol, or4-hydroxyphenethlyl alcohol (FIG. 7B).

In another embodiment, the compounds that increase FXR-mediatedtranscription and/or increase or potentiate EcR-mediated transcriptioncomprise insecticide synergists. In a further embodiment, theinsecticide synergists that increase FXR-dependent transcription, and/orincrease or potentiate EcR-mediated transcription, comprise piperonylbutoxide (PB), seasamin, sesame oil, piperine, myristicin, or apiole(FIG. 7C).

In an embodiment, the compounds that increase FXR-mediatedtranscription, and/or increase or potentiate EcR-mediated transcription,comprise monoterpenes. In a further embodiment, the monoterpenes thatincrease FXR-dependent transcription, and/or increase or potentiateEcR-mediated transcription, comprise tea tree oil (terpenen-4-ol,1,8-cineole, and α-terpineol), carvacrol, thymol, perillyl alcohol,fenchyl alcohol, or pinane diol (Table 4 and FIGS. 8A and 8B).

The compounds that increase FXR-mediated transcription, and/or increaseor potentiate EcR-mediated transcription, may also comprise diterpenes.In a further embodiment, the diterpenes that increase FXR-dependenttranscription, and/or increase or potentiate EcR-mediated transcription,comprise forskolin, 1-trans-Δ⁹-tetrahydrocannabinol (THC), abietic acid,croton oil, and other phorbol-like diterpenes such as phorbol12,13-dibutyrate, mezerein, and also ingenol 3,20-dibenzoate, cafestol,kahweol, or their acetate derivatives.

Also, the compounds that increase FXR-mediated transcription, and/orincrease or potentiate EcR-mediated transcription, may comprisetriterpenes. In yet a further embodiment, the triterpenes that increaseFXR-dependent transcription, and/or increase or potentiate EcR-mediatedtranscription, comprise essential oils from myrrh and frankincense,β-boswellic acid, oleanolic acid, rosemary oil, or 20α- or20R-hydroxycholesterol.

The present invention also comprises compound that can suppress theactivity of FXR or EcR. Compounds that inhibit the ecdysone receptor(EcR) may suppress FXR activity. Also, the compounds that inhibit FXRactivity may suppress FXR activity promoted by FXR modulators.

In one example embodiment, 1 μM cucurbitacin D (cue D) may suppressFXR-dependent activity promoted by JH III and chenodeoxycholate (CDCA)(40 μM each), 7-fold and 58-fold, respectively (FIG. 8C). Also, theΔ¹-unsaturated congener cucurbitacin I may inhibit farnesol-inducedactivity with an IC₅₀˜50 nM (data not shown).

The compounds that increase FXR-mediated transcription, and/or increaseor potentiate EcR-mediated transcription, may also comprisefurocoumarins or phenylpropanoids. In one example embodiment, thefurocoumarins and phenylpropanoids that increase FXR-dependenttranscription, and/or increase or potentiate EcR-mediated transcription,comprise the furocoumarins, bergamot oil and bergamotin (from Earl Greytea) (FIG. 8D), myristicin, or apiole, or the phenylpropanoid,methyleugenol (FIG. 8E).

In other embodiments of the present invention, the compounds thatincrease FXR-mediated transcription and/or increase or potentiateEcR-mediated transcription may comprise coumarins and flavonoids. Thecoumarins and flavonoids that increase FXR-dependent transcription may,for example, comprise silybin, tangeretin or a rotenonic acid (FIG. 8F)or 8-prenylnaringenin and isozantholhumol from hops.

The compounds that increase FXR-mediated transcription, and/or increaseor potentiate EcR-mediated transcription, may also comprise linoleicacid metabolites. In yet a further embodiment, the linoleic acidmetabolites that increase FXR-dependent transcription, and/or increaseor potentiate EcR-mediated transcription, comprise cis-jasmone or methyljasmonate.

Also, in an embodiment, the compounds that increase FXR-mediatedtranscription and/or increase or potentiate EcR-mediated transcriptioncomprise polyketides from hops. In yet a further embodiment, thepolyketides that increase FXR-dependent transcription, and/or increaseor potentiate EcR-mediated transcription, comprise humulone (FIG. 8G).

Also, in an embodiment, the compounds that increase FXR-mediatedtranscription and/or increase or potentiate EcR-mediated transcriptioncomprise xanthines. In yet a further embodiment, the xanthines thatincrease FXR-dependent transcription, and/or increase or potentiateEcR-mediated transcription, comprise theophylline, caffeine, 8-Br-cAMP,dibutyryl cAMP, or 8-Br-cAMP in combination with theophylline (FIG. 8H).

In an embodiment, man-made insecticides may increase FXR-mediatedtranscription and/or increase or potentiate EcR-mediated transcription.For example, in an embodiment, man-made insecticides that increaseFXR-mediated transcription, and/or increase or potentiate EcR-mediatedtranscription, comprise cinerins, pyrethrins, jasmolins, syntheticpyrethroids including cypermethrin, permethrin, phenothrin, andbioallethrin (Table 5).

TABLE 5 FXR Activation by Plant Essential Oils and Ingredients DOSE FXRACTIVITY* PLANT OIL FXR ACTIVITY INGREDIENT (μM) (fold-induction)allspice 7 balm 4 balsam fir 3 juvabione basil 10 juvocimene 25 9bergamot 10 bergamotin 25 10 bergapten 100 1 black pepper N.T piperine50 4 cardomom 3 cassia bark 1 cedarwood 9 α-ionone, β-ionone 150 10clove 3 eugenol 200 1 coffee N.T caffeine 3000 12 theophylline 3000 12caffeic acid 100 1 cafestol 20 12 kahweol 20 10 coleus forskholi N.Tforskolin 10 >100 cottonseed 1 croton 3 ingenol-3,20-dibenzoate 10 5derris N.T rotenone 20 1 rotenonic acid 20 20 Echinacea 7 echinoloneN.T. Fennel 7 Frankincense 30 β-boswellic acid 25 11 Ginger 4 Hops 188-prenylnaringenin 20 38 Humulone 20 8 Xanthohumol 1 Isoxanthohumol 20 9Lupulone 20 1 milk thistle 4 silybin 100 10 myrrh 22 nutmeg N.T.methyleugenol 250 4 olive oil 4 phenethyl alcohols 400 4 orange N.T.limonene 300 1 perillyl alchol 300 4 origanum 6 carvacrol 300 6 thymol300 6 parsley N.T. myristicin 250 4 safrole 250 1 pyrethrum 10 pyrethrinN.T. cinerin N.T. jasmolin N.T. rice N.T. γ-tocotrienol 20 1 sage 8sesame 3 sesamin 100 12 sesamol 1 spruce N.T. abietic acid 50 23 teatree 8 1,8-cineole 400 2 α-terpineol 402 terpinen-4-ol 400 2 thyme 13vetiver 24 ylang ylang 17 cis-jasmone 1000 6 methyl jasmonate 1000 18*FXR activity is defined as the ratio of FXR-dependent CAT activityattained in appropriately transfected cells at maximal doses of plantoil (or indicated doses of ingredients) over that for the vehicle.Activity level of “1” indicates no increase in activity over vehicle.

Also, insecticides such as o,p-DDT (but not p,p-DDT), chlordane, kepone,lindane, dieldrin, toxaphene, aroclor 1254,2,3,7,8-tetrachlorodibenzo-p-dioxin, malathion, diazinon, chlorpyrifos,parathion, ethion, chlorfenapyr, pyrethrin, permethrin, fenvalerate, orfipronil may increase FXR-mediated transcription and/or increase orpotentiate EcR-mediated transcription in an embodiment of the presentinvention (Table 6; FIG. 9).

TABLE 6 FXR Is Activated by Synthetic Insecticides DOSE FXR ACTIVITYCOMPOUND STRUCTURE (μM) (fold-induction) chlordane

 5 7 o,p-DDT

 5 3.5 dieldrin

 5 17 tetrachlorodibenzo-p- dioxin

  0.1 15 malathion

50 13 diazinon

50 33 phosdrin

100  1 pyrethrin I (pyrethrum extract)

Maximum 10 permethrin

25 5 fenvalerate

25 9

The present invention recognizes that small doses of ecdysone may primethe EcR-dependent transcriptional response provoked by JHs. Ecdysonesmay stabilize a conformation of EcR that permits JHs to bind moreeffectively. Since JH potentiators may affect the maximaltranscriptional response elicited by ecdysone, this may be similar toallosteric enzymes whose effectors alter the apparent V_(max) withoutchanging the K_(m) value. Thus, the variable cytotoxicities provoked bydietary or ectopically-applied ecdysones, JHs, or insecticides indifferent insects may be uniquely imparted by theirpharmacologically-distinguishable EcR/USP complexes.

In one embodiment, the present invention describes the use of JHantagonists as compound that may be used to modulate insect growth. Inone embodiment, the JH antagonists may increase FXR-mediatedtranscription. Alternatively, the JH antagonists may inhibitFXR-mediated transcription.

For example, instead of acting as an antagonist, precocene I, a polardimer of precocene (Rf=0.27; m/z=380)(FIG. 10A), and its 6,7-dimethoxycongener precocene II (both from Sigma-Aldrich), and a polar dimer ofprecocene (FIG. 10A), may induce FXR-dependent activity, but withreduced potencies (EC₅₀=150 μM) compared to farnesol. Precocene I can beinactive in RAR, RXR, PPAR, or GR-based transcriptional assays, whichsuggests that it may be relatively specific for FXR (data not shown).Also, FXR may be activated by other precocene-like JH antagonists (FIG.10C) including 3,4-dimethoxy-6-isopentenylphenol (3-fold induction at100 μM) and a tricyclic dichromene (29-fold induction at 25 μM), di-,tri-, and tetraprenylated ubiquinones (U2, U3, and U4) (FIG. 10F).

In contrast, FXR may be inhibited by precocene metabolites andderivatives, such as 6,7-dihydroxy precocene and 6,7-dihydroxy coumarin(esculetin), and ubiquinone-1 (10B, 10C, and 10D).

Thus, in one embodiment, transcriptional activity programmed bymuristerone-primed ecdysone receptors may be potentiated by juvenilehormones, compounds derived from food sources, and insecticides (FIG.11). The transcriptional effects may, in some embodiments, requireligand binding domain sequences of the EcR or FXR (FIG. 12). In anembodiment, mutations in the LBD of FXR or EcR may be utilized toprepare constructs specific to various types of potential insecticidecompounds.

Also, natural JH's may increase FXR-mediated transcription, and/orincrease or potentiate EcR-mediated transcription. For example, FXR mayrespond to the JH I analog ZR354 in which the ethyl groups of JH I ethylare replaced by similarly bulky dimethyl groups.

In one embodiment, the natural JH's that increase FXR-mediatedtranscription, and/or increase or potentiate EcR-mediated transcription,comprise all “natural” JHs, e.g., JH 0, JH1, and JH II (where ethylgroups are substituted for JH III methyl groups) (FIG. 13).

D. Compositions for Use as Insecticides

Compounds of the present invention may be used in the form ofcompositions and can be applied to the crop and/or plant to be treated,simultaneously with, or in succession with, other compounds such asfertilizers, micronutrient donors or other preparations which influencethe growth of plants. The compounds can also be selectively combinedwith herbicides, as well as, other insecticides, fungicides,bactericides, nematocides, molluscicides or mixtures of several of thesepreparations and, if desired together with further carriers, surfactantsor application promoting adjuvants employed in the art of formulation,and as described in U.S. Pat. Nos. 6,737,383, 6,630,465, 6,586,470,6,603,044, 6,617,341, 5,942,542, and 5,849,320.

For example, when applying the compound of the present invention, thecompound may be applied in a form as it is without adding other activecomponents. When the compound of the present invention is applied forplant protection purpose, the compound can be prepared into generaltypes of formulations for plant protection use, such as wettable powder,granules, dust, emulsifiable concentrate, water soluble powder,suspension concentrate, flowable liquid, and the like.

The inert carrier used in this invention may be either solid or liquid.Where the compound of the present invention is prepared into a solidformulation, appropriate additives and carriers may be incorporated withthe compound. The solid carrier may be a solid such as soybean flour,cereal flour, wood flour, bark flour, saw dust, powdered tobacco stalks,powdered walnut shells, bran, powdered cellulose, extraction residues ofvegetables, powdered synthetic polymers or resins, clays (e.g. kaolin,bentonite, and acid clay), talcs (e.g. talc and pyrophillite), silicapowders or flakes (e.g. diatomaceous earth, silica sand, mica and whitecarbon, activated carbon, powdered sulfur, powdered pumice, calcineddiatomaceous earth, ground brick, fly ash, sand, calcium carbonatepowder, calcium phosphate powder and other inorganic or mineral powders,chemical fertilizers (e.g. ammonium sulfate, ammonium phosphate,ammonium nitrate, urea and ammonium chloride), and compost. Thesecarriers may be used alone or as a mixture thereof.

Where the compound of the present invention is prepared into a liquidformulation, an appropriate solvent may be used for dissolving ordispersing the compound in the liquid type formulation. The liquidcarrier is that which itself has solubility or which is without suchsolubility but is capable of dispersing an active ingredient with theaid of an adjuvant. The following are typical examples of the liquidcarrier and can be used alone or as a mixture thereof: water; alcoholssuch as methanol, ethanol, isopropanol, butanol and ethylene glycol;ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone,diisobutyl ketone and cyclohexanone; ethers such as ethyl ether,dioxane, dipropyl ether and tetrahydrofuran; aliphatic hydrocarbons suchas kerosene and mineral oils; aromatic hydrocarbons such as benzene,toluene, xylene, solvent naphtha and alkylnaphthalenes; halogenatedhydrocarbons such as dichloroethane, chloroform, carbon tetrachlorideand chlorobenzene; esters such as ethyl acetate, diisopropyl phthalate,dibutyl phthalate and dioctyl phthalate; amides such asdimethylformamide, diethylformamide and dimethylacetamide; nitriles suchas acetonitrile; and dimethyl sulfoxide. In one embodiment, thecomposition of the invention may also applied to the plant foliage orplant stem or insect habitat as a dilute spray prepared from any of theabove-said formulations.

Also, to provide uniformity and stability to the compound in theprepared compositions, it is possible to add surface active agents intoeach formulation upon necessity. To emulsify, disperse, dissolve and/orwet an active ingredient, a surfactant is used. There is no limitationfor the surface active agent, and examples of the surface active agentthat can be added to the above-mentioned formulations include nonionicsurface active agents, such as polyoxyethylene-added alkyl ether,polyoxyethylene-added higher fatty acid ester, polyoxyethylene-addedsorbitan higher fatty acid ester and polyoxyethylene-added tristyrylphenyl ether, a sulfate ester of polyoxyethylene-added alkyl phenylether, an alkyl benzene sulfonate, a polycarbonate, a lignin sulfonate,a formaldehyde condensate of alkyl naphthalene sulfonate, and acopolymer of isobutylene and maleic anhydride.

Further, to stabilize the dispersion of an active ingredient, tackify itand/or bind it, there may be used adjuvants such as casein, gelatin,starch, methyl cellulose, carboxymethyl cellulose, gum arabic, polyvinylalcohols, turpentine, bran oil, bentonite and ligninsulfonates. Also, toimprove the flowability of a solid product, there may be used adjuvantssuch as waxes, stearates and alkyl phosphates. Adjuvants such asnaphthalenesulfonic acid condensation products and polycondensates ofphosphates may be used as a peptizer for dispersible products. Adjuvantssuch as silicon oils may also be used as a defoaming agent. For example,adjuvants such as those described in U.S. Pat. No. 5,942,542 may beused.

While the compound of the present invention may be used alone, it can becombined for the use with one or more of various types of other plantprotection chemicals, for example, fungicides, insecticides, acaricidesand synergists. Also, in one embodiment, the composition may comprise aseed coating, formulated as described in U.S. Pat. No. 5,849,320. Theinsecticide compounds of the present invention may be used in admixturewith other agricultural and horticultural disease or pest controllers,acaricides, nematicides, bioagrochemicals, etc.; and herbicides, plantgrowth regulators, manures, etc. depending upon scenes using the presentagricultural and horticultural insecticides, in order to expand bothspectrum of controllable diseases and insect pest species and the periodof time when effective applications are possible or to reduce thedosage.

The insecticide compounds of the present invention may be applied usinga variety of protocols. Thus, the composition may be applied to a cropon which the insect pests are expected to appear, or to a site where theappearance of the insect pests is undesirable. The insecticidecompositions of the present invention may also be applied to the plantseeds or the cultivation mediums for seeding such as soil to be seeded,the mat for raising seedlings, water, and the like, by the method ofapplication to a nursery box, seed powdering, etc. or by the method ofseed disinfection. For controlling the pest insects generated on fruittrees, cereals, upland field for vegetables, etc., it is also possibleto make a plant absorb the compounds of the present invention by a seedtreatment such as powder coating, dipping, etc., irrigation intoseedling-raising carrier such as seedling-raising vessel, or plantinghole, or by treatment of the culture solution for water culture.

The applied dosage of the insecticide compounds of the present inventionmay be varied depending upon various factors such as the insect pests tobe controlled, the growth state of a plant, weather, environmentalconditions, the preparation form, application method, application siteand application time. In one example embodiment, the does may comprise arange of 0.1 g to 10 kg (in terms of the active ingredient) per 10 acresdepending upon purposes. Thus, the amount of an active ingredient ineach of the composition may be in a range of from 0.01 to 90% by weight,or preferably from 0.05 to 85% by weight based on the total weight ofthe formulation. In dusts, granules, or emulsifiable concentrates, thesuitable content thereof is from 0.01 to 50% by weight. Each of theprepared formulations, such as wettable powder, emulsifiableconcentrate, suspension concentrate and flowable solution, may bediluted with water or other solvent to be prepared and adjusted into thesuspension or emulsion with a desired concentration to be applied tocrop plants. For the formulations, such as granular and dustformulations, the formulation itself is directly applied to the targetcrop plants or soil.

The compositions of the present invention comprise compounds thatincrease FXR-mediated transcription and/or increase or potentiateEcR-mediated transcription present as non-toxic doses. JH levels in theinsect support the use of FXR and EcR-activating compounds asinsecticides. Thus, in alternate embodiments of the present invention,the compounds may comprise a dosage ranging from 0.01 μM to about 10 mM,or from about 0.1 μM to about 1 mM, or from about 0.5 μM to about 50 μM.

For example, JH III circulates in honeybee hemolymph at 0.5 μM(Elekonich, M. M. et al., 2001, J. Insect Physiol., 47:1119-1125), whichmatches the concentration of the JH III precursor farnesyl diphosphatefound in the rat liver (Bruenger, E. and H. C. Rilling, 1988, Anal.Biochem., 173:321-327). Also, JH III titers in Diploptera hemolymph are6 mM during the middle of the gonotrophic cycle and 10-fold lower atother times (To be, S. S., et al., 1985, Experientia, 41:1028-1034).Purification of 1.6 mg of JH I (M_(r)=294) from 875 Cecropia abdomens(380 g) translates to 1.5 μM in the whole insect (Roller, H. and K. H.Dahm, 1968, Recent Prog. Horm. Res., 1968, 24:651-80). These amounts arenear the doses of JH III or farnesol (2 μM) that elicit FXR-dependentactivity in the CHO assay of the present invention.

EXAMPLES Example 1 Materials and Methods

A. Cell Growth Conditions

Chinese hamster ovary (CHO K1) cells were grown in Dulbecco's modifiedEagle medium: nutrient mixture F-12 (1:1) containing 5% fetal bovineserum and supplemented with 50 u/ml penicillin, and 50 μg/mlstreptomycin (Life Technologies) in a water jacketed incubator held at37° C. and maintained with a 5% CO₂ atmosphere.

B. Chemicals

Chemicals were purchased from Sigma-Aldrich Chemical Company (St. Louis,Mo.) unless noted. Plant oils were manufactured by Aura Cacia(Weaverville, Calif.). Juvocimene was synthesized as described (Mestres,R. and E. Munoz, 1996, Synthetic Comm., 26:1309-1319). Myristicin,apiole, bergamotin, tangeretin, bisabolol, and cucurbitacin wereobtained from Indofine Chemical Company (Somerville, N.J.).Methyleugenol was provided by the Batelle Chemical Company (Columbus,Ohio). Man-made insecticides were purchased from Chem Service (WestChester, Pa.). The precocene-like suicide substrate3,4-dimethoxyisopentenylphenol was prepared as described Bowers, W. S.,et al., 1976, Science, 217:647-648) except that a 3,4-dimethoxylatedreactant was used.

C. Transfection Assay

The transcriptional assay utilized for the study of EcR potentiation byJH (FIGS. 2-5) was modified from the assay described by Forman et al.,(Forman, B. M., et al., 1995, Cell 81, 687-695) as described below.Cells were seeded in 6-well polypropylene culture plates (Falcon) with10⁵ cells per well on the day prior to transfection. Transfection wassubsequently performed using either calcium phosphate (Kitareewan, S.,et al., 1996, Mol. Biol. Cell, 7:1153-1196) or a GenePorter reagent(Gene Therapy Systems, Inc; San Diego, Calif.) following manufacturer'sprotocols. Each well received 1.25 μg of (EcRE)₅-ΔMTV-CAT (five copiesof the hsp27 EcRE inserted into an mouse mammary tumor virus (MTV)promoter upstream of the chloramphenicol acetyltransferase (CAT) gene)or (EcRE)₅-ΔMTV-LUC (the same promoter attached to firefly luciferase),1.25 μg of pCH111(SV40 early promoter linked to the β-galactosidasegene) to normalize CAT activity, and 0.25 μg of each expression plasmid(EcR, FXR, RXR, USP) that was tested. The cells were incubated withplasmid DNA for seven hours and then washed with 1×PBS. Muristerone A(murA; Alexis Biochemicals) or 20-hydroxyecdysone (20E; Sigma) dissolvedin ethanol to a concentration of 10 mM was diluted as necessary to thefinal assay concentration (FAC) in 2 ml of fresh incubation medium thatwas then applied to the cells. Similarly, JHIII (Sigma) was dissolved indimethyl sulfoxide (DMSO) to a concentration of 80 μM and diluted intothe incubation medium to its final assay concentration (20, 40, 80, 160μM). For experiments to test the effects of chenodeoxycholate (CDCA,Sigma) on FXR and EcR, CDCA was dissolved in DMSO at 20 mM and dilutedin the culture medium to a final concentration of 20 μM. A correspondingvolume of ethanol and DMSO were added to control cells for allexperiments. For all experiments, the cells were allowed to incubatewith the medium for 24 hours before collection and cell lysates wereprepared by described methods (Kitareewan et al., 1996). Bothβ-galactosidase and CAT reporter activity were measured based onpreviously used methods (Kitareewan et al, 1996). Luciferase assaysusing luciferin followed the specifications of the manufacturer.

Transcriptional activity, measured as ¹⁴C-chloramphenicol counts (formeasuring CAT activity) or relative luciferase activity (RLU) wasquantified for each cell lysate. The counts were then normalized byadjusting for differences in β-galactosidase activity, sinceβ-galactosidase expression is controlled by a constitutive promoter andprovides an estimate of cell mass. Data were normalized asfold-induction based on differences in reporter gene activity betweenhormonally treated and control cells

For the analysis of the ability of various compounds to activate FXR(FIGS. 6-13), the same assay was used with the rat FXR and mouse RXRα.DNAs were added to cells for 7 hours and subsequently washed with PBS.Activators were then added in fresh DMEM-F12 media containing 5%charcoal-adsorbed FBS and incubated at 37° C. for 22 hours. CAT andβ-galactosidase activities were measured from cell lysates prepared bythree cycles of freeze-thawing as described (Kitareewan, S. et al.,1996, Mol. Biol. Cell, 7:1153-66).

D. Chromatographic Analysis of Compounds

Thin layer chromatography to analyze the chemical structure of thefarnesoids and other compounds tested for their ability to activate FXRand/or activate or potentiate EcR was carried out using Whatman LK6Dsilica gel plates (60 Å, 250 μm). The mobile phase was 90% hexane/10%ethyl acetate. For the separation of precocene I, 10 milligrams ofmaterial was applied to a 20 cm×20 cm plate. Ultraviolet light-absorbingmaterial in three zones was scraped from the plate, eluted withchloroform:methanol

(1:1), dried, and resuspended in methanol. A portion of the material wasderivatized with trimethyl silyl chloride and subjected to gaschromatography-mass spectrometry as described (Kitareewan, S. et al.,1996, Mol. Biol. Cell, 7:1153-66). Samples were assayed forFXR-dependent transcriptional activity as outlined above.

Example 2 Description of Plasmid Vectors

Expression plasmids encoding Drosophila EcR (CMX-EcR), Drosophila USP(CMX-USP), rat FXR (CMX-FXR), human RXRα (CMX-hRXRα), glucocorticoidreceptor (CMX-GR), and a glucocorticoid receptor trans-activating domainfused to an ecdysone DBD and LBD (CMX-GEcR) have been described (Yao etal., 1992, Cell, 71: 63-72; Yao et al., 1993, Nature 366:476-478; andForman, B. M., 1995, Cell, 81: 687-693). The expression plasmids areconstructed by inserting restriction fragments continuing theappropriate coding sequence for the gene to be expressed into the CMXPL1plasmid. For hRXRα, an EcoRI fragment of human RXR-α (hRXRα) wassubcloned into the CMXPL1 plasmid.

The FXR expression vector (pRS-rFXR) contains DNA sequences encoding aconstitutively active promoter derived from the Rous sarcoma virus longterminal repeat (LTR) fused to the complementary DNA sequence encodingthe rat farnesoid-X-activated receptor (Forman et al., Cell 1995; NCBIAccession No. U18374) which is followed by a DNA sequence specifying theSV40 polyadenylation signal. Briefly, the expression plasmid pRS-rFXRwas derived from pRS-hGR as follows: pRS-hGR was digested with Kpn I andBam HI restriction endonucleases to release DNA sequences specifying thehuman glucocorticoid receptor (GR) and cDNA encoding rat FXR and its 5′and 3′ untranslated sequences was then inserted into Kpn I and Bam HIdigested pRS-hGR.

GGF, comprising the glucocorticoid (G) amino terminus and DBD linked tothe LBD of FXR, was prepared following a strategy similar to that usedto construct GGEc (KS Christopherson, et al., 1992, Proc Natl Acad SciUSA, 89:6314-6318), where the GR amino terminus and DBD (appending 23amino acids downstream from the conserved gly-met) were fused to the FXRLBD. To construct GGF, the plasmid pRShGR_(nx) (Giguere, V. et al.,1987, Nature, 330:624-9), with Not I and Xho I sites flanking the GRDBD, was first used as a template in a polymerase chain reaction (PCR)assembled with the forward primer 5′-GGAATGATTGCATCATCGATAAAATTCG-3′(Cla I restriction site underlined) (SEQ ID NO: 1) and the reverseprimer 5′-GAGGTCTCGAGTGAGACTCCTGTA-3′ (Xho I site underlined) (SEQ IDNO: 2). Cla I- and Xho I-digested pRShGR_(nx) was then ligated to the132 bp PCR product. The resulting plasmid was digested with Xho I andBam HI and the GR-containing DNA fragment was isolated and ligated to anXho I-Bam HI LBD fragment from an FXR variant prepared by hybridizingthe oligonucleotide 5′-CTCGAGTGTATGTATACAGGTTTGTTAACTGAA-3′ (SEQ ID NO:3) to another oligonucleotide 5′-AACAAACCTGTATACATACACTCGA-3′ (SEQ IDNO: 4) which was then ligated to a Hpa I-digested fragment from thepRSV-FXR expression vector. The DBD/LBD junction in GGF is5′-GMNLEARKTKKKIKGIQQATTGVSQECMYTGLLTEIQCKS-3′ (SEQ ID NO: 5) where theGR residues are underlined, amino acids GM are the last two residues ofDNA binding domain and amino acids from FXR are in bold (notunderlined).

The GGEc vector (Christopherson et al., 1992, Proc. Natl. Acad. Sci.,USA, 89:6314) is derived from the rat GR expression vector pRSV.GGG(Miesfeld et al., 1986, Cell, 46:389) and contains the Rous sarcomavirus LTR fused to DNA encoding the rat glucocorticoid receptor (GR)amino terminus and DNA binding domain fused to a DNA sequence encodingthe Drosophila melanogaster ecdysteroid receptor (EcR) (NCBI AssessionNo. M74078; Koelle, M. R., et al., 1991, Cell 67:59-77) ligand bindingdomain (LBD). In GGEc, rat GR LBD amino acids 528 to 795 were replacedby EcR LBD amino acids (EcR amino acids 329 to 878).

The GEcEc (i.e., GRdEcR) vector has been described previously (Yao, T.P, et al., 1993, Nature 366: 476-479; No, D., et al., 1996, Proc. Natl.Acad. Sci USA, 93:3346-3351). Briefly, GEcEc was constructed by ligationof a Not I-Bam HI fragment containing the DBD and LBD of a modified EcRcDNA, EcR_(nx), in place of the DBD and LBD of the similarly modified GRexpression vector construct pRShGRnx (Giguere et al., 1987, Nature 330:624). The modified EcR cDNA was constructed using site-directedmutagenesis (Kunkel, 1985, Proc. Natl. Acad. Sci., USA, 82:488) toinsert Not I (oligonucleotide template:5′-CCTGCGCCACGGCGGCCGCCGGAGCTGTGCCTG-3′) (SEQ ID NO: 6) and Xho I(oligonucleotide template: 5′-GTGGGTATGCGCCTCGAGTGCGTCGTCCC-3′) (SEQ IDNO: 7) sites immediately flanking the DBD. This results in conversion ofamino acids 259-261 from ValGlnGlu to ArgProPro and amino acid 331 fromPro to Leu.

Reporter plasmids EcR₅-ΔMTV-LUC, and MMTV-LUC have also been described(Yao et al., 1992, Cell, 71: 63-72; Yao et al., 1993, Nature366:476-478; and Forman, B. M., 1995, Cell, 81: 687-693). The reporterplasmids may be constructed by inserting a ecdysone response element(e.g., 5′-GATCCGACAAGGGTTCAATGCACTTGTCA-3′; SEQ ID NO: 8) at position−77 of a mouse mammary tumor virus (MMTV or MTV) promoter-reporter geneconstruct, such as MTV-CAT, MTV-LUC, MTV-GFP (Christopherson, K. S.,1992, Proc. Natl. Acad. Sci., USA, 89: 6314-6318). The (EcRE)₅-ΔMTV-LUCconstruct was produced by subcloning the promoter region of MTV into themultiple cloning site of the p-LUC plasmid (Promega); this vectorresembles the (EcRE)₅ΔMTV-LUC described previously (No et al, Proc.Natl. Acad. Sci, USA, 93:3346-3351).

Construction of the three Drosophila EcR isoform vectors (EcRA, EcRB1,EcRB2) has been described (Mouillet, J-F, et al., 2001, Eur. J. Biochem.268:1811-1819). To generate the vectors EcR-A, B1, and B2 sequences(Koelle et al., 1991, Cell: 67, 59-77; Talbot et al., 1993, Cell:73:1323-1337; NCBI Accession Nos. S63761 and S63762) generated by PCRwere cloned into the BamHI and XbaI sites of the pcDNA3 vector(InVitrogen). The EcRA DNA fragment was produced by using PCRamplification of EcRA DNA with the pWT57 vector as a template (Talbot etal, 1993). The forward primer, DEAf(5′-CACCCGGATCCACCATGTTGACGACGAGTGGACAA) (SEQ ID NO: 9) was used withthe reverse primer, DEr (5′-ACCTCTCTAGACTATGCAGTCGTCGAGTGGTC) (SEQ IDNO: 10) to produce a fragment that was subsequently digested with BamHIand XbaI and ligated into a pcDNA3 vector digested with BamHI and XbaI.The plasmid encodes a version of EcRA that includes 849 amino acids, anddeletes the F domain. The EcRB1 DNA fragment was produced by using PCRamplification with the pMK1 vector (Koelle et al, 1991; Talbot et al,1993) using the aforementioned DEr primer with the forward primer, DEB1f(5′-CACCCGGATCCACCATGAAGCGGCGCTGGTCGAAC) (SEQ ID NO: 11). The fragmentwas subsequently digested and cloned into the pcDNA3 vector as describedfor A. The plasmid encodes a version of EcRB1 that includes 878 aminoacids, and deletes the F domain. The EcRB2 DNA fragment was produced byusing PCR amplification with the pWT56 vector (Talbot et al, 1993) usingthe aforementioned DEr primer with the forward primer, DEB2f(5′-CACCCGGATCCACCATGGATACTTGTGGATTAGTA-3′) (SEQ ID NO: 12). Thefragment was subsequently digested and cloned into the pcDNA3 vector asdescribed for A. The plasmid encodes a version of EcRB2 that includes669 amino acids, and deletes the F domain. The VP16dEcR vector has alsobeen described previously (Yao, T. P, et al., 1993, Nature 366: 476-479;No, D., et al., 1996, Proc. Natl. Acad. Sci USA, 93:3346-3351; Mouilletet al., 2001; Henrich et al., 2003). The VP16 EcR vector was constructedusing PCR amplification with pWT57 with the reverse primer, Der(described above), together with the forward primer Def(5′-CACCCGGATCCACCATGAAGAAGGGACCTGCGCCA-3′) (SEQ ID NO: 13). Thefragment was subsequently digested with BamHI and XbaI and cloned in tothe multiple cloning site of pVP16 (Clontech). The resulting vectorencodes a protein consisting of the VP16 activation domain linked to theC-E domain of EcR and consisting of 626 amino acids.

The VP16CfUSP vector is also previously described (Palli, S R, et al.,2003, Eur J. Biochem, 270:1308-15). The USP vector contains the USP LBDof Choristoneura fumeriferana (NCBI Accession No. AF045891). Both VP16chimeras contain an N-terminal domain that is active in mammalian cells(Louvion et al, 1993, Gene:131:120-134). To generate VP16CfUSP, PCRamplification with a forward primer including an EcoRI site and areverse primer including a BamHI site was used to produce a fragmentthat encodes the D-F domains of Choristoneura fumiferana USP (Accession#AAC31795). The resulting PCR product was digested with EcoRI and BamHIand cloned into the pVP16 vector (Clontech) to produce the fusionprotein. Other VP16-USP fusion vectors VP16-dUSPF1, F2, and F3, havebeen generated. These vectors were constructed using the pZ7-1 vector(Henrich et al, Nucl. Acids Res, 18:4143-4148, 1990) encoding Drosophilamelanogaster USP. F1 was constructed with the forward primer5′-TTTTGAATTCAGCGGCAGCAAGCACCTCTGC-3′ (SEQ ID NO: 14) together with thereverse primer, 5′-TTTTAAGCTTTAGAGTCGGGACCCTACTCC-3′ (SEQ ID NO: 15).The resulting PCR product was digested with EcoR1 and HindIII and clonedinto pVP16 (Clontech) digested with EcoR1 and HindIII. The resultingprotein encodes a VP16 activation domain fused to the last six aminoacids of the A/B domain and the C-E domains of USP. F2 was constructedusing the same pZ7-1 vector, reverse primer and the forward primer(5′-TTTTGAATTCTGCTCTATTTGCGGGGATCGG-3′) (SEQ ID NO: 16). The resultingPCR product was cloned into VP16 using the same approach described forF1. The resulting protein encodes a VP16 activation domain fused to theC-E domain of Drosophila melanogaster USP. F3 was constructed as both F1and F2 except that the forward primer used was(5′-TTTTGAATTCAAGCGCGAAGCGGTCCAGGAG'3′) (SEQ ID NO: 17). The resultingprotein encodes a VP16 activation domain fused to the D-E domain ofDrosophila melanogaster USP.

Example 3 JHIII Potentiates Ecdysteroid-Induced Transcriptional Activityin a Mammalian Cell Line Transfected with EcR

In an initial series of studies, a GRdEcR chimera that consists of therat glucocorticoid receptor (GR) activation domain attached to the EcRDBD and LBD (FIG. 1) was cotransfected along with mRXRα into CHO cells.The response of transfected cells to murA was measured using a(EcRE)₅-ΔMTV-CAT reporter plasmid that carries five tandem repeats ofthe hsp27 EcRE linked to the MTV (mouse mammary tumor virus) promoterand the chloramphenicol acetyltransferase gene (CAT).

Results of a typical experiment are shown in FIG. 2. Cotransfection withGrdEcR and RXR evoked a detectable response at dosages as low as 0.1 μMmurA. It was found that juvenile hormone III (JHIII) potentiated theresponse of murA in a dose-dependent manner (using 20, 40, 80, and 160μM JHIII) at submaximal murA dosages (0.1 μM and 1 μM murA) (FIG. 2,sets 2 and 3, respectively). JHIII did not display the ability to evokea response that was greater than the maximal level induced by 10 μMmurA. Despite the structural resemblance between the LBDs of EcR and thevertebrate FXR, which is highly responsive to JHIII alone (Forman etal., 1995), JHIII alone did not show an effect on transcription mediatedby the GEcEc chimera (FIG. 2, set 1). Thus, this experiment shows thatalthough JH by itself may not be able to evoke a EcR mediated response,JH can potentiate the effect of EcR ligands.

Example 4 Ecdysteroid Responsiveness and JHIII Potentiation in EcRChimeras Depends Upon Activating Ligand and Heterodimeric Partner

The potentiation experiments were repeated with a chimera encoding theVP16 activation domain connected to the DBD and LBD of Drosophila EcR togenerate the construct VP16dEcR (FIG. 3). Luciferase was used as areporter gene, (EcRE)₅-ΔMTV-LUC. All activities are normalizedfold-inductions in relative light units from cells incubated withhormone for 20 hours compared to luciferase activity levels in cellsincubated with solvents only. For all combinations, 80 μM JHIII wasused. The dosage of muristerone A (murA) used was based upon preliminaryexperiments to determine a submaximal dosage wherein a JHIII effect, ifany, was detectable. All data are based on the mean normalizedfold-inductions from at least three replicates. The range (coefficientof variation) of fold-inductions was less than 15% among replicates.

VP16dEcR tested with muristerone A generated a sensitive and robustecdysteroid response (based on normalized fold-induction). The VP16dEcR,partnered with mouse RXR (mRXR), showed a response to 0.01 μM and 0.1 μMmuristerone A that was further potentiated by JHIII (FIGS. 3 a and 3 b,respectively). The VP16dEcR chimera also displayed a discernibleresponse to 20E at 10 μM (over 20-fold) using RXR. The VP16dEcR/20Eactivity was only minimally affected by the additional presence ofJHIII, however (not shown).

The VP16dEcR was also tested with VP16CfUSP as the heterologous bindingpartner. FIG. 3 c shows results at 0.1 μM murA. The same degree ofpotentiation was observed using the VP16dEcR/USP combination as with theVP16dEcR/RXR when using murA, except that a higher murA dose wasrequired to achieve the same efficacy. The normalized level ofJHIII-mediated potentiation of the murA response with USP was similar tothat seen for RXR. However, the combination of VP16dEcR with VP16CfUSPcombination showed no response to 10 μM 20E and was not affected by theadditional presence of JHIII (data not shown).

Example 5 Drosophila EcR Isoforms Display Different Capabilities inMammalian Cells that Depend Upon Ligand and Heterodimeric Partner

In order to evaluate the activity of the various EcR isoforms, each ofthe three natural Drosophila melanogaster EcR isoforms, (EcRA, EcRB1,and EcRB2) were cotransfected into CHO cells with the VP16CfUSP fusionprotein. Results are shown in FIG. 3, sets d-f. Again, for allcombinations, 80 μM JHIII was used, and the dosage of muristerone A(murA) used was based upon preliminary experiments to determine asubmaximal dosage wherein a JHIII effect, if any, was detectable. Forthe results shown in FIG. 3, all data are based on the mean normalizedfold-inductions from at least three replicates, except for EcRA (tworeplicates). All activities are normalized fold-inductions from cellsincubated with hormone for 20 hours compared to luciferase activitylevels in cells incubated with solvents only. Each fold inductionrepresents an average based on three or four replicates, and range wasless than 15% mean fold-inductions for each data point. All combinationswere also tested with JHIII alone (80 μM), which registered nosignificant effect on normalized RLU activity.

The different EcR isoforms are known to differ at the N-terminal regionof the protein, which is the part of the protein involved indimerization of EcR with either USP, RXR or other appropriate partners.All three isoforms showed some transcriptional capability in themammalian cell culture system. Interestingly, however, none of theisoform constructs showed the same dosage sensitivity seen withVP16dEcR/RXR (direct comparison not shown).

For example, when tested in the absence of hormone, the EcRB1/VP16CfUSPcombination showed a relatively high level of ligand-independenttranscription. Thus, the EcRB1/VP16CfUSP had between 10 and 20-foldhigher basal levels than any other EcR construct tested. The EcRAisoform in combination with VP16CfUSP also showed a level of basal levelof transcription that was 2 to 3 fold higher than that observed forEcR-B2. In contrast, the basal activity of the B2/VP16CfUSP dimer wasabout the same as the basal activities produced by the VP16dEcR/RXR andGEcEc/RXR (i.e., GRdEcR/RXR).

FIG. 4 shows the effects of murA, 20E, and JHIII on RLU activity inducedby (EcRE)₅ΔMTV-LUC in CHO cells cotransfected with a Drosophila EcRisoform and VP16CfUSP where sets 1, 2, and 3 in the figure correspond toEcRA, EcRB1 and EcRB2, respectively. When tested with VP16CfUSP, allthree Drosophila isoforms were induced by about 30-40 fold at 1 μM murA.The response of all EcR isoforms in the presence of VP16CfUSP waspotentiated by the presence of 80 μM JHIII in the presence of 0.1 μMmurA (FIG. 4). It was found that the effect was dose-dependent (data notshown) similar to the results seen with GEcEc (i.e, GRdEcR). The rangeof the normalized fold inductions for each experiment was found to varyby less than 15% for each experiment.

The Drosophila EcR isoforms and VP16CfUSP were also tested with 20E(FIG. 4). At a dosage of 10 μM 20E, all three constructs generated aconsistent and discernible transcriptional response. Only theEcRB2/VP16CfUSP dimer (FIG. 4, set 3) was potentiated significantly bythe additional presence of JHIII, however. That only the B2 EcR isoformis potentiated by JHIII in the presence of 20E indicates that JHIIIpotentiation may depend upon both the N-terminal domain of EcR and theactivating ecdysteroid.

The activity observed among the three EcR isoforms was dependent uponthe identity of the heterologous partner used. For example, in contrastto the robust response observed when RXR was tested with VP16dEcR, RXRdid not mediate a response to murA as a dimer with either EcRA or EcB2.Only EcRB1/RXR displayed a response to murA among the three isoforms(FIG. 3 d, e, and f), but the levels of transcription were relativelylow and dramatically reduced from those noted for the B1/USPcombination. The murA response was further potentiated by JHIII with theB1/RXR combination, though JHIII by itself failed to evoke any response.Unlike the 20E response noted for all the isoforms with USP as apartner, none of the isoforms showed a 20E response with RXR as aheterodimeric partner. Absolute transcription levels were alsorelatively low with RXR.

The results indicate that ligand-independent and ligand-dependenttranscription as well as JHIII potentiation may depend upon an interplayof the EcR N-terminal domain, the activating ecdysteroid, and theheterodimeric partner. Thus, it was found that responsiveness of thenatural EcR isoforms to 20E requires USP (rather than RXR) as a dimericpartner. Also, among the three isoforms (EcRA, EcRB1, and EcRB2), JHIIIpotentiation in the presence of 20E occurred only with the EcRB2 isoformand USP. Specific combinations of the EcR N-terminal domain and theheterodimeric partner (e.g. VP16 and RXR, B2 and USP) result in afunctional receptor that is capable of showing an ecdysteroid responseand/or JHIII potentiation. Levels of ligand-independent transcriptionalso depend upon both the EcR N-terminal domain and the heterodimericpartner. The potentiation observed in the experiments cannot beattributed to the activation of RXR by either JHIII or a JHIIImetabolite, since JHIII showed no activity by itself on the assays(Harmon, M. A., et al., 1995, Proc. Natl. Acad. Sci. USA, 92:6157-6160;Saez, E., et al., 2000, Proc. Natl. Acad. Sci. USA, 97:14512-14517).

Example 6 Assay of FXR Interaction with USP

It is known that the FXR/RXR heterodimer may respond to JHIII. Thus, aseries of experiments were carried out to determine the ability ofinsect USP to mediate an ecdysteroid and/or JHIII response inconjunction with FXR.

The combination of FXR and USP evoked a low level response in CHO cellsto JHIII (FIG. 5). The response observed is attributable to endogenousexpression of low levels of RXR in these cells (data not shown). Theaddition of ecdysteroids (murA or 20E) with JHIII induced no elevationof FXR-mediated activity (FIG. 5). Also, USP was unable to potentiate aresponse to 20 μM CDCA (Chiang et al, 2000, J. Biol. Chem.,275:10918-10924), the strongest activator of FXR known to date. Inaddition, EcR was unresponsive to CDCA alone or as a potentiator of murAresponse (data not shown). These experiments indicate that whereas EcRis able to interact with USP or RXR, and FXR interacts with RXR, FXRdoes not interact with USP to induce transcription. For FIG. 5, allactivities are normalized log (fold inductions) based on RLU activityfrom a simultaneous run that was repeated twice and generated similartrends, though dosage levels varied.

Example 7 Evaluation of Compounds for Activity at the Farnesoid Receptor(FXR)

In these experiments, candidate juvenoids were tested for their abilityto activate FXR mediated transcription by transfecting Chinese hamsterovary (CHO) cells using the FXR plasmid, mouse RXRα, and theΔMTV-(EcRE)₅-CAT reporter as described herein. An RXR-dependentCRBPII-CAT reporter plasmid was employed in parallel assays to discernactivators specific for this receptor.

A. FXR Responds to Endogenously-Produced Farnesol Metabolites

Farnesoid-like molecules define a metabolic pathway that begins withfarnesyl diphosphate (Weinberger, C., 1996, TEM, 7:1-6). Thus, in theseexperiments, endogenously-produced metabolites of farnesol (FIG. 6) (50μM FAC) were assayed as FXR effectors. FIG. 6 shows the metabolic routefrom farnesyl diphosphate (FDP) to methyl farnesoate in mammals orjuvenile hormone III in insects and the relative efficacy of eachisoprenoid as an inducer of FXR-dependent transcription. A negative signindicates no activity and positive signs correlate with the efficacy(ratio of maximal inducible activity achieved with the highestnon-cytotoxic dose of activator compared to that obtained with vehicle).Transcriptional activities ranged from 2-fold increases for farnesoicacid to 20-fold inductions for juvenile hormone III when tested at 50μM.

Thus, nerolidol induced FXR-dependent CAT activity with a potency(EC₅₀=15 μM) and efficacy (9-fold induction) like its farnesol isomer(FIG. 6). Alcohol and aldehyde dehydrogenases oxidize farnesol farnesoland farnesoic acid (Christopher, J. and G Popjak, 1961, J Lipid Res.,2:244-257), which exhibited 2 to 3-fold activity increases in FXRmediated transcriptional activity, respectively. Insects and mammalstransform farnesoic acid into methyl farnesoate (Stoolie, D. A. and F CBaker, 1985, Juvenile hormone biosynthesis. In: Comprehensive InsectPhysiology, Biochemistry, and Pharmacology Edited by G A Kerkut, L IGilbert, vol. 7. pp. 363-389. Oxford: Pergamon Press; 1985: 363-389)which showed a 6-fold induction of FXR mediated transcription. Methylfarnesoate is epoxidized in insects to the FXR activator JH III, whichinduced CAT activity 15-fold. Thus, it was found that FXR respondsvariably to all endogenously-produced open chain sesquiterpenoidmetabolites of farnesyl diphosphate in the biochemical pathway thatextends from farnesol to JH III.

B. Juvenile Hormone Mimetics Induce FXR-Dependent Transcription

FXR-activating farnesoids have been described as JH agonists in insectbioassays (Schneiderman, H. A., and L I Gilbert, 1964, Science,143:325-333). Like farnesol, nerolidol was effective as a juvenoid andas an FXR effector (Table 4). The chlorophyll metabolite phytolincreased activity three times, thereby evincing marginal JH activity(Table 4). In contrast, neither the monoterpenes linalool (200 μM) norgeraniol were effective as FXR activators or juvenoids.

Synthetic juvenoids (Table 4 and FIG. 7A) were also evaluated. The ethylester of 7,11-dichloro-2-ene farnesoic acid (ZR232) (Law, J. H. et al.,1966, Proc. Natl. Acad. Sci. USA, 55:576-578), induced CAT activity5-fold when added at 50 μM (FIG. 7A). In addition, the syntheticjuvenoids, methoprene and pyriproxyfen, increased FXR-dependent activitywith efficacies like that produced by JH III (Table 4 and FIG. 7A).These results indicate that FXR activates transcription in response toisoprenoids and chemicals previously reported to have insect JHactivities.

Plant-derived JH agonists were also examined as FXR effectors.Farnesol-like echinolone (FIG. 7A), an essential oil of echinacea(Jacobson, M., et al., 1975, Lloydia, 38:473-476) maximally inducedFXR-dependent activity 7-fold. Juvocimene (found in sweet basil) andjuvabione (from balsam fir) (FIG. 7B), two JH mimetics WS (Bowers, W. S.et al., 1966, Science, 154:1020-1; Bowers, W. S, and R Nishida, 1980,Science, 209:1030-1032), increased RXR mediated transcription 10-foldand 3-fold, respectively. Synthetic juvocimene (25 μM) increasedFXR-dependent activity 5-fold. Also, α-bisabolol (50 μM), an analog ofjuvabione found in chamomile, increased activity 13-fold (FIG. 7B).

Certain olive oil vehicles have been distinguished as juvenoids ininsect molting assays (Carlisle, D. B. and P. E. Ellis, 1968, Science,162:1393-1394). Extra-virgin olive oil or its redolent tyrosine-like 2-,3,-and 4-hydroxyphenethyl alcohol constituents (400 μM) all elevatedFXR-dependent activity 3-fold. Unsubstituted phenethyl alcohol was inertat this dose. Activation of FXR by natural and synthetic JHs offersfurther evidence that FXR has functional attributes of an insect JHreceptor.

C. FXR is Activated by Insecticide Synergists

The observation that sesame oil and its sesamin and sesamoliningredients increased the toxicity of insecticides led to thedevelopment of synergist analogs like piperonyl butoxide (PB) (FIG. 7C)and the description of these compounds as JH agonists (Bowers, W. S.,1968, Science, 161:895-7). PB and sesamin (100 μM) induced FXR-dependentactivity 9-fold and 12-fold, respectively. Crude sesame oil (Sigma)maximally increased activity 3-fold; three commercial brands of toastedsesame oil were more effective, however, delivering maximal activitiesbetween 8-fold and 17-fold. Also, 4-fold induction was produced by 50 μMpiperine, a PB analog from black pepper, as well as by myristicin andits p-dimethoxylated congener apiole in dill and parsley (FIG. 7C) (bothat 250 μM FAC). The sesamolin cleavage product sesamol (FIG. 7C) wasinactive at 400 μM FAC as were PB analogs with small side chains such aspiperonyl alcohol, piperonylic acid, and safrole. These results indicatethat FXR shares functional qualities similar to those expected of aninsect JH receptor, and that the synergistic actions of PB and sesaminmay partly reflect intrinsic JH activities.

D. FXR is a Target for Plant Secondary Metabolites

Commercially available oils from various plants were also examined asFXR effectors. For example, cedarwood oil and its redolent sesquiterpeneα- and β-ionone isomers increased FXR-dependent activity with potenciesand efficacies like those induced by farnesol (Table 5). It is knownthat sesamin blocks liver HMG CoA reductase activity, that farnesol isanti-proliferative, and that FXR is inhibited by thehypocholesterolemia-inducing plant steroid guggulsterone (Wu, J. et al.,2002, Mol Endocrinol., 16:1590-7; Urizar, N. L. et al., 2002, Science,296:1703-6). Given the reported biochemical activities for sesamin,farnesol, and FXR, phytochemicals previously reported to modulate plasmacholesterol levels or growth in mammals were tested as FXR effectors.The data are summarized by molecular class in Table 5.

i. Monoterpenes: Given that FXR responds to endogenously-producedisoprenoids, plant-derived monoterpenes were examined for activity. Teatree oil, which contains insecticidal terpinen-4-ol (40% of mass),1,8-cineole (eucalyptol), and α-terpineol, maximally elevatedFXR-dependent CAT activity 8-fold (Table 5 and FIG. 8A). Each of theseconstituents induced activity only two-fold at 800 μM, but notably 400μM of any pairwise mixture increased activity like tea tree oil itself(FIG. 8A). Carvacrol and thymol (300 μM) in oregano inhibit tumor cellgrowth (Case, G. L. et al., 1995, Lipids, 30:357-9; Burke, Y. D. et al.,1997, Lipids, 32:151-6) and both induced CAT activity 6-fold (Table 5).Limonene in orange oil blocks tumors, inhibits HMG CoA reductase, and isbeing tested as a human chemotherapeutic agent (Crowell, P. L. and M. N.Gould, 1994, Crit. Rev. Oncog., 5:1-22; Elegbede, J. A. et al., 1984,Carcinogenesis, 5:661-4; and McNamee, D., 1993, Lancet 342, 801).Limonene and limonene oxide (400 μM) were inactive in activation of FXR,but either enantiomer of its metabolite perillyl alcohol inducedFXR-dependent CAT activity 4-fold (Table 5). This dose matches thatrequired to inhibit cell growth in culture (He, L. et al., 1997, J.Nutr., 127:668-674). Finally, while menthol and fenchone (fennel) wereinactive (0.5 mM), 0.5 mM fenchyl alcohol induced activity 5-fold, asdid 0.5 mM pine tree-derived pinane diol (Table 5).

ii. Diterpenes: Forskolin, one of the most robust FXR effectors yetdescribed (Howard, W. C. et al., 2000, Tox. Appl. Pharm., 163:195-202),increased activity more than 100-fold when added at 2 μM. Likeforskolin, 1-trans-Δ⁹-tetrahydrocannabinol (THC) from the cannabis plantcontains a tricyclic ring. In addition to their psychoactive effects,cannabinoids block cell growth, inhibit DNA synthesis, and lower theincidence of spontaneously-arising mouse liver tumors (Carchman, R. A.,et al, 1976, Cancer Res., 36:95-100; Munson, A. E. et al, 1975, JNCI,55:597-602). A 5-fold increase in FXR-dependent CAT activity waselicited by 15 μM THC, but not by its Δ⁸-isomer or by cannabinol (Table5). Abietic acid (100 μM), another forskolin-like diterpene in ginko,spruce, and fir, induced CAT activity 50-fold (Table 5). FXR-dependentactivity was also increased 3-fold by tumor-promoting croton oil at thehighest non-cytotoxic dose. Phorbol-like diterpenes from croton tigliumand related plants such as phorbol 12,13-dibutyrate, mezerein, andingenol 3,20-dibenzoate, increased activity 2, 3, and 5-fold,respectively, when added at 10 μM (Table 5). Phorbol and ingenol wereinactive.

The palmitate esters of cafestol and kahweol have been identified as themediators of the hypercholesterolemic effects of coffee in humans (butnot in other primates or rodents) (see e.g., Weusten-Van der Wouw, M. P.et al., 1994, J Lipid Res., 1994, 35:721-735). Cafestol, kahweol andtheir acetate derivatives (20 μM) induced FXR-dependent CAT activitybetween 10- and 20-fold (FIG. 8B and Table 5). At this dose, the majorditerpenoid-fatty acyl ester in coffee oil, cafestol palmitate, wasinert.

iii. Triterpenes.

Resin from Commiphora molmol (myrrh) exhibits insecticidal activity inLepidoptera (Shonouda, M. L. et al., R M Farrag, O M Salama, 2000, JEnviron Sci Health B, 35:347-56). FXR was activated by essential oilsfrom myrrh and frankincense with maximal inductions approaching 20 timesmore than vehicle (Table 5). Some of the FXR-dependent activity promotedby frankincense is derived from triterpenoid components, β-boswellicacid and oleanolic acid (25 μM), which increased activity 11- and3-fold, respectively (Table 5). Ursolic acid is a rosemary ingredientrelated to oleanolic acid that inhibits mouse skin tumors (Huang, M. T.et al., 1994, Cancer Res., 54:701-708; Nishino, H. et al., 1988, CancerRes., 48:5210-5215). While rosemary oil maximally induced FXR-dependentactivity 12-fold (Table 5), ursolic acid (50 μM) was ineffective as wereits polyketide constituent rosemarinic acid and its curcumin congener.

Cucurbitacins are phytoecdysteroids that inhibit ecdysone receptor (EcR)function (Dinan, L. et al., 1997, Biochem J., 327:643-50). CucurbitacinD (1 μM) suppressed FXR-dependent activity promoted by JH III and CDCA(40 μM each), 7- and 58-fold, respectively (FIG. 8C). The Δ¹-unsaturatedcongener cucurbitacin I also inhibited farnesol-induced activity with anIC₅₀˜50 nM (data not shown). The 22-oxo-Δ²³-ene group of cucurbitacin iscritical for EcR antagonist activity, allegedly by forming covalentadducts between its α,β-unsaturated carbonyl group and amino acids inthe EcR ligand binding domain (Dinan, L. et al., 1997, Biochem J,327:643-50). 22R-Hydroxycholesterol is metabolized in mammals topregnenolone via a 20,22-dihydroxycholesterol intermediate with anapparent K_(m) of 7 μM (Sugano, S., et al., 1966, J Biochem (Tokyo)1996, 120:780-7). Coincidentally, FXR-dependent transcription wasinduced 10-fold by 20α- or 22R-hydroxycholesterol (7.5 μM), but not by22S-hydroxy-, 7-keto-, or 7α-hydroxycholesterols (data not shown). It ispossible that the 20,22-dihydroxy group can be metabolized to the22-oxo-Δ²³-ene functionality in insects and mammals to generate EcR orFXR antagonists.

iv. Furocoumarins and phenylpropanoids. The Earl Grey tea flavoringbergamot oil and its constituent bergamotin, which possesses a geranylgroup, both induced CAT activity 8-fold when tested at 25 μM (Table 5and FIG. 8D). The unprenylated bergamot ingredients bergapten(5-methoxypsoralen) and its hepatocarcinogenic 8-methoxylated psoralencongener were inactive at 50 μM (FIG. 8D). Also, methylenedioxyphenylslike myristicin and apiole in dill and parsley weakly elevated CATactivity 4-fold at 250 μM, but their congener safrole, a rodent livercarcinogen (Miller, E. C. et al., 1983, Cancer Res., 43:1124-34), wasinert (FIG. 8E). While the alkenylbenzenes eugenol and caffeic acid werealso inactive, methyleugenol, a multi-site rodent carcinogen (Johnson,J. D., et al., J. Agric. Food Chem. 2000, 48:3620-3632) found in nutmegand other plants, induced FXR-dependent activity 4 times more thanvehicle at this same dose (FIG. 8E).

v. Coumarins and flavanoids. The flavolignin silybin from milk thistle(silymarin) induces macromolecular synthesis in the hepatectomizedrodent liver (Fausto, M. and J. Sonnenbichler, 1977, Hoppe-Seyler's Z.Physiol. Chem., 358:141-147), arrests cells in the G₁ phase of the cycle(Zi, X. and R. Agarwal, 1999, Proc. Natl. Acad. Sci. USA, 96:7490-7495),and shows anti-proliferative effects (Katiyar, S. K. et al., 1997, J.Natl. Cancer Inst., 89:556-66). Silybin increased FXR-dependent activity18-fold at 50 μM (Table 5). While silymarin lowers cholesterol betterthan silybin (Krecman, V. et al., 1998, Planta Med., 64:138-42), FXRactivity was maximally increased only 4-fold by the former (data notshown). FXR may respond to other analogs of silybin in milk thistle suchas silydianin, silychristin or their metabolites. Taxifolin is onesilymarin component that has been shown to inhibit HMG CoA reductaseactivity in hepatocytes (Theriault, A., et al., 2000, J. Lipid Res.,41:1969-1979). FXR was unresponsive to taxifolin or to other flavonoidssuch as genistein, quercetin, catechins, and gossypetin (50 μM).However, activity was increased 4-fold in response to the same dose oftangeretin, a methoxylated flavone in citrus fruits.

The insecticidal actions of the derris plant flavonoid-like componentrotenone allegedly result from its ability to inhibit electron transportand respiration (Chance, B., and G. Hollunger, 1963, J. Biol. Chem.,278:418-431). Rotenone also reduces the incidence ofspontaneously-arising liver tumors in male mice. These findings animateda test of rotenone as an FXR effector. Rotenone was inactive (FIG. 8F),but its rotenonic acid derivative with a cleaved furan ring, inducedFXR-dependent activity 20-fold when added at 20 μM (FIG. 8F). This mayhave some functional significance since metabolites of rotenone such as6′,7′-dihydro-6′,7′-dihydroxyrotenone and 8′-hydroxyrotenone, which arehydroxylated in the proximity of the furan ring, can be generated usingmicrosomal homogenates prepared from either insect or rodent tissues(Fukami, J. I. et al., 1967, Science, 155:713-6).

vi. Linoleic acid metabolites. An oil extract of ylang ylang, whichemits a jasmine-like aroma, maximally increased FXR-dependent activity17-fold (Table 5). One of its components, cis-jasmone, a linoleic acidmetabolite and defensive signal that is released by plants followingherbivore damage (Birkett, M. A. et al., 2000, Proc. Natl. Acad. Sci.USA, 97:9329-9334), elicited a 6-fold increase in activity when added at1 mM (Table 5). An identical dose of jasmonic acid had no effect, but a17-fold increase was produced by its methyl ester (Table 5). Due totheir volatilities, it is anticipated that the high doses of jasmonoidsrequired for FXR activation in cell culture may not correspond to thedoses that mediate attractant, repellant, or insecticidal activities inthe animal.

vii. Polyketides. Since an extract of hops maximally inducedFXR-dependent activity 18-fold, some individual components were tested.The flavanone 8-prenylnaringenin and its methylated isomerisoxanthohumol (20 μM) elicited 38- and 9-fold increases in activity,respectively (FIG. 8G and Table 5). However, FXR was not activated byxanthohumol, the chalcone precursor to isoxanthohumol that is producedduring beer brewing (FIG. 8G). Humulone increased activity 8-fold, butlupulone, a structurally-related hops ingredient with an additionalisoprenyl group, was inert (Table 5, both tested at 20 μM).

xiii Xanthines. FXR activation by forskolin animated tests of otherplant compounds that modulate cAMP levels. FXR-dependent activity wasinduced 12-fold by 3 mM theophylline or caffeine, but congeners such astheobromine, hypoxanthine, xanthine, adenine, and the cAMP metabolite5′-AMP were ineffective (FIG. 8H and data not shown). Modest (4-fold)increases in CAT activity were afforded by 8-Br-cAMP or dibutyryl cAMP(1 mM). However, mixing theophylline (3 mM) with 8-Br-cAMP (1 mM)increased activity more than 100-fold like forskolin itself (data notshown). The FXR-activating theophylline dose matches its concentration(4 mM) in tobacco hornworm larvae three days after eating tomato leavessprayed with 1% theophylline, a dose that reduces leaf consumption byhalf (Nathanson, J. A., 1984, Science, 226:184-7). Given that caffeineand theophylline are present in coffee beans and tea leaves atconcentrations that kill Manduca larvae, xanthines may function asnatural insecticides (Nathanson, J. A., 1984, Science, 226:184-7).

In summary, the foregoing experiments indicate that FXR activatestranscription in response to a broad range of plant secondarymetabolites, which were previously described as insecticides or asmodulators of cholesterol or growth in higher metazoans.

E. FXR is Activated by Diverse Class of Man-Made Insecticides

FXR activation by plant-derived JHs and secondary substances provokedtests of man-made insecticides. Its sodium channel-based toxicitynotwithstanding (Soderlund, D. M.,: 1985, Neurotoxicology, 6:35-46), apyrethrum extract of chrysanthemum flowers maximally inducedFXR-dependent activity 13-fold. Candidate active ingredients are thestructurally-related cinerins, pyrethrins, and jasmolins since FXR wasinduced by synthetic pyrethroids (25 μM) including cypermethrin (15-foldinduction), permethrin (5-fold induction), phenothrin (8-foldinduction), and bioallethrin (14-fold induction) (Table 6).

Organochlorine insecticides (5 μM) such as o,p-DDT (but not p,p-DDT),chlordane, kepone, lindane, dieldrin, and toxaphenes increased CATactivity 3, 7, 12, 5, 17, and 9-fold, respectively (data not shown).Other organochlorines like aroclor 1254 (5 μM) and2,3,7,8-tetrachlorodibenzo-p-dioxin (100 nM) increased activity 5 and15-fold, respectively (data not shown). FXR was activated byorganophosphates such as malathion, diazinon, chlorpyrifos, andparathion (25 μM) with potencies and efficacies like farnesol (Table 6).Others like ethion and coumaphos were more efficacious and more potent,exhibiting 50- and 16-fold increases in activity, respectively, whentested at 5 μM. Lower molecular weight insecticides phosdrin, carbaryl,and imidan were inactive. Phenylpyrazoles such as chlorfenapyr whichlowers ATP levels and structurally-related fipronil that distinctivelyblocks chloride channels increased FXR-dependent activity 10- and20-fold, respectively, at 25 μM (FIG. 9). FXR was unaffected byimidacloprid, a nicotine-like compound that interferes withacetylcholine receptor function. These results indicate that FXR mayactivate transcription in response to structurally-diverse syntheticchemicals that manifest pleiotropic cytotoxicities through equallydisparate mechanisms.

F. FXR may be Inhibited or Activated by a Metabolite of the JHAntagonist Precocene

Chromene ring-containing precocenes are plant-derived JH antagoniststhat hasten insect metamorphosis (Bowers, W. S. et al., 1976, Science,193:542-7). Like safrole and other alkenylbenzenes, the precocenes arerodent hepatocarcinogens (Wiseman R. W. et al., 1987, Cancer Res.,47:2275-83). Their alkylation of DNA and proteins and their metabolismto 3,4-diols hinted that precocenes may form reactive epoxides (Brooks,G. T., et al., 1979, Nature, 281:570-572; Pratt, G. E., et al., 1980,Nature, 284:320-323). To more firmly establish a role for FXR as afunctional homolog of an insect JH receptor, precocene could was testedfor its ability to function as an FXR antagonist. Instead of acting asan antagonist, precocene I and its 6,7-dimethoxy congener precocene II(both from Sigma-Aldrich) induced FXR-dependent activity 15-fold, butwith reduced potencies (EC₅₀=150 μM) compared to farnesol. Precocene Iwas inactive in RAR, RXR, PPAR, or GR-based transcriptional assays,which suggests that it may be relatively specific for FXR (data notshown).

Different lots of precocene induced FXR-dependent activity with varyingefficacies, prompting further analysis by thin layer chromatography(TLC). UV-absorbing material eluted from silica was tested for CATactivity and analyzed gas chromatography and mass spectrometry followingtrimethylsilane derivatization. Non-polar species corresponding toprecocene I (R_(f)=0.44; m/z=190) did not affect FXR (FIG. 10A; Polar),but a more polar dimer (R_(f)=0.27; m/z=380) increased activity 22-fold.Its structure was inferred from the observation that FXR was activatedby 5,11-dimethyltetrahydrochrysene, but not by its 6,12-dimethylcongener (FIG. 5A) (Meyers, M. J. et al., 1999, J Med Chem 1999,42:2456-68). The most polar species (R_(f)<0.16) elevated activity12-fold and had molecular weights consistent with hydroxylatedprecocenes (FIG. 10A). These likely arose by silica gel-catalyzed airoxidation since they were not detected by GC/MS in the crude sampleprior to TLC. Precocene I (25 mg, Sigma-Aldrich, 99% purity) wasseparated by thin layer chromatography using hexane-ethyl acetate (9:1)as the mobile phase. Aliquots (1× and 3× doses) were tested for FXReffector activity in parallel. In FIG. 10A, precocene carbons arenumbered for reference, and the putative structure for the precocenedimer is presented based upon FXR responsiveness (box within graph). Oneprospective silica gel-catalyzed air oxidation product of precocene(3,4-dihydroxyprecocene) is depicted.

Given that FXR did not respond to the 3,4-diol or its precursor epoxide(data not shown) and that insects and mammals produce other hydroxylatedspecies, experiments were performed to determine whether precocene couldbe metabolized to some other FXR effector. Incubations with mouse livermicrosomes yielded ethyl acetate-extractable material that induced FXRactivity 3-fold, which hinted at the presence of some liver activatorssuch as farnesol or bile acids (FIG. 10B). In these experiments, NADPHand either DMSO and 100 μM precocene I were mixed with CD-1 mouse livermicrosomes and incubated at 37° C. for one hour. Reaction products wereextracted with ethyl acetate, dried, resuspended in DMSO, and tested forFXR effector activity. Parallel microsome incubations with 100 μM ofTLC-purified inert precocene I (R_(f)=0.44; m/z=190) generated ethylacetate-soluble products that reduced this activity by 70%. Sinceprecocene II is O-demethylated (Soderlund, D. M. et al., 1980, J. Agric.Food Chem., 1980, 28:724-731) and 6,7-methylenedioxyprecocene (FIG. 10C)is not a JH antagonist (Bowers, W. S., 1969, Toxicology of theprecocenes. In: Insecticide Mode of Action Edited by JR Coats. New York:Academic Press; 1969), it was surmised that the 6,7-catechol is the FXRantagonist produced by microsomes. FIG. 10C shows that precocene I andprecocene II are metabolically interconverted by methylations anddemethylations; also shown is a model (in box) for oxidation (O) andtautomerization of 3,4-dimethoxy-6-isopentenylphenol to a quinonemethide and subsequent adduct formation with cellular nucleophiles.Support for this conjecture came from the finding that the FXR activityinduced by farnesol was inhibited 44% by 100 μM esculetin, an analog ofprecocene with a similarly positioned 6,7-catechol (FIG. 10D). In theseexperiments, increasing amounts of esculetin were added along with 45 μMfarnesol to CHO cells transfected with plasmids that express FXR andmouse RXRα, along with a ΔMTV-(EcRE)₅-CAT reporter plasmid. NormalizedCAT activities are expressed as mean values±standard deviationcalculated from triplicate well samples. The ineffectiveness ofesculetin and its analog 7-hydroxy-6-methoxycoumarin as FXR agonists andthe ability of 7-methoxycoumarin (all at 1 mM) to increase activity10-fold emphasize the specificity of congeners. FXR was activated byother precocene-like JH antagonists (FIG. 10C) including3,4-dimethoxy-6-isopentenylphenol (3-fold induction at 100 μM) and atricyclic dichromene (29-fold induction at 25 μM). It has been proposedthat, like preocene, these suicide substrates disrupt metamorphosis bycovalently binding to nucleophilic DNA or proteins following theiroxidation to epoxides, catechols, or quinone methides (Bowers, W. S. etal., 1976, Science, 217:647-648). Since P450 metabolism may be impairedin cultured cells, it is not entirely unexpected to find that precoceneand analogs did not inhibit FXR-dependent activity in the CHO cell-basedassay.

Additional experiments were performed to determine whether analogs ofprecocene function as potential endogenously-produced FXR antagonists.Ubiquinone-1 (U1) with a single isoprene unit is a congener ofdecaprenylated U10 (coenzyme Q), which functions in electron transport.At 10 μM, U1 completely inhibited farnesol-induced FXR-dependentactivity (FIG. 10E). In contrast, di-, tri-, and tetraprenylatedubiquinones (U2, U3, and U4) increased activity 7, 5, and 3-fold,respectively (data not shown). FXR activation by U2 is depicted (FIG.10F). U6, U8, U9, and U10 were inactive (data not shown). U2, U3, and U4are detected in bacteria and U6 is found in yeast (Daves, G. D. et al,1967, Biochemistry, 6:2861-2866). However, only U9 and U10 have beenreported in insects and mammals (Olson, R. E., 1966, Vitum Horm.,24:551-74). U1, U2, U3, and U4 have not been measured in mammals andhence their physiological significance is not known. Nonetheless, theseresults illustrate how in situ-generated electrophilic metabolites ofprecocene may antagonize the effects of JHs via interactions with an FXRhomolog in insects.

Example 8 Ecdysone Receptor Activity is Potentiated by JHs andInsecticides

The activation of FXR by natural and synthetic JHs and its inhibition byprecocene indicated that FXR may have pharmacological features of along-postulated insect JH receptor. The prominent candidate for thisJH-responsive macromolecule in insects is the structurally-relatedecdysone receptor (EcR). Like FXR, EcR heterodimerizes with RXR andbinds to the hsp27 ecdysone-responsive DNA element (Forman et al.,1995), but distinctively activates transcription in response tomuristerone A (MurA or MUR), a synthetic ecdysone (Yao, T. P., et al.,1992) Cell, 71:63-72). Despite these similarities, a MurA-induciblechimeric receptor (GEcEc), constructed by fusing the humanglucocorticoid receptor (GR) amino-terminus to the Drosophila EcR DNAand ligand binding domains, failed to respond to 80 μM JH III (FIG.11A). In the experiments shown in FIG. 11A, muristerone A (MUR) wasadded at the indicated doses in ethanol vehicle, and increasing amountsof JH III (12, 25, or 50 μM) were added (underlying triangles). Numbersover bars indicate the ratio of the GEcEc-dependent activity produced by50 μM JH III in the presence of the indicated dose of MUR to theactivity produced by MUR alone. Relative light units (RLU) from fireflyluciferase in cell lysates are expressed relative to renilla luciferase(Promega).

Given that JHs modulate ecdysone actions, it was originally anticipatedthat JH III might antagonize MurA inducible GEcEc-dependent signaling.In contrast, and as discussed above, the GEcEc-dependent transcriptionalactivity induced by MurA was increased 3-times more by the addition of80 μM JH III (FIG. 11A). The effect was seen with as little as 0.5 nMMurA, an amount of MurA that barely elevated activity by itself. SimilarJH III-mediated increases were afforded by higher doses of MurA (1 and10 nM, FIG. 11A). Thus, farnesol (45 μM) elicited a 4-fold increase inactivity over that provided by MUR alone. In the experiments shown inFIG. 11B, CHO cells were separately transfected with plasmid DNAs thatexpress mouse RXRα or GEcEc (1.25 μg plasmid DNA per well), ortransfected with both plasmids or none. MUR was added to cells at 10 nMand JH III at 50 μM. Normalized CAT activity was determined by measuringβ-galactosidase activity produced by cotransfected SV40-β-gal plasmidDNA. Note that the ordinate axis is broken. The potentiative effect wasalso seen using a VP16-Chironomus Usp substituted for its mammalianhomolog RXR (data not shown). Also, both RXR and GEcEc were essentialfor activity (11B).

Given that EcR responds to farnesol and JH III, other FXR-activatingnatural and synthetic JHs and insecticides were tested. For theexperiments shown in FIG. 11C, CHO cells were transfected with rat FXRand mouse RXRα or GEcEc and mouse RXRα as described in Methods.Muristerone A (0.2 μM) was added alone or with 10 or 20 μM juvocimene(J) to GEcEc-transfected cells. Farnesol (45 μM) or juvocimene was addedto FXR-transfected cells. Mean CAT activity is displayed±standarddeviation from triplicate well samples. Juvocimene, sesamin, andpiperonyl butoxide (25 μM) potentiated the MUR-inducible GEcEc-dependentactivity between two and four times (FIG. 11C and data not shown).

Also, GEcEc-dependent activity was induced between two and three timesby other insecticides (25 μM) including diazinon, endosulfan (5 μM),coumaphos, permethrin, and precocene, while 25 μM chlorpyrifospotentiated the MUR response nine times (FIG. 11D). For the experimentsshown in FIG. 11D, triplicate wells of CHO cells were transfected withGEcEc and RXR, after which muristerone A (MUR) was added at 10 nM inethanol vehicle. Cells were incubated with the indicated natural andsynthetic insecticides (25 μM), except endosulfan which was added at 5μM. Plus and negative signs refer to MUR addition. Numbers above barsindicate the ratio of activity from a lysate of cells treated withinsecticide plus MUR divided by that treated with MUR alone.

Although FXR and EcR exhibit substantial homology, FXR was unresponsiveto muristerone A, ecdysone, or 20-hydroxyecdysone. Reciprocally, the FXRactivator chenodeoxycholic acid (CDCA) did not induce GEcEc (data notshown). Thus, it appears that EcR and FXR may both activatetranscription in response to natural and synthetic JHs and insecticides,but that the two receptors may not be seamlessly interchangeable.

Example 9 JH and Insecticide Effects Mediated Via FXR and EcR LigandBinding Domains

The ligand binding domains (LBDs) of nuclear receptors map to theircarboxyl termini (Kumar, R. and E. B. Thompson, 1999, Steroids,64:310-9). To ask whether JHs and insecticides transduce their effectsvia their putative LBDs, chimeric receptors that link the GR DNA bindingdomain (DBD) to the carboxy-terminal regions of FXR or EcR wereexamined.

EcR/GR Chimeras

For these experiments, the luciferase reporter construct was used. Toobviate influences from other transcription factors, results werenormalized using a reporter plasmid from which CAT expression is drivenby a minimal promoter consisting of dimerized GREs linked upstream of a13 base pair TATA box DNA element derived from the adenovirus E1B gene.

The MUR-inducible GR/EcR hybrid, GGEc, that fuses the rat GR aminoterminus and DBD to the Drosophila EcR LBD (Christopherson, K. S., etal., 1992, Proc Natl Acad Sci USA, 89:6314-6318) induced transcriptionbetween two and four times more with 50 μM farnesol, its isomernerolidol, or diazinon than with 0.2 μM MUR alone (FIG. 12A). In thisexperiment, GGEc and RXR were transfected into CHO cells with either aCAT or a luciferase reporter plasmid containing 1.5 kilobase pairs ofthe glucocorticoid-inducible mouse mammary tumor virus (MTV) promoter.Farnesol was added at 25 μM with or without 0.2 μM muristerone A.Normalized CAT activities are mean±standard deviation as measured fromlysates sampled from triplicate wells. Like GEcEc, this activityrequired an RXR-expressing plasmid (data not shown).

As seen for FXR, GGEc responded to juvocimene (20 μM), the JH mimeticfrom basil, by increasing activity 7 times more than MUR (FIG. 12B). Inthese experiments, CHO cells were transfected with a GGEc-expressingplasmid, a mouse RXRα expression plasmid, and an adenovirus E1bTATA-(GRE)₂-CAT reporter plasmid. The GGEc plasmid was eliminated fromDNA mixtures added to cells transfected in parallel. CAT activities weremeasured from lysates of cells from triplicate wells treated withethanol vehicle, juvocimene (20 μM), muristerone A (0.2 μM), or both.

Functional harmony for FXR and EcR was underscored by the observationthat the FXR effector ubiquinone-2 (10 μM) similarly increasedGGEc-dependent activity 7 times more than MUR (FIG. 12C). In theseexperiments, CHO cells were transfected with DNAs as described above forthe experiment shown in 12B, but ubiquinone-2 (10 μM) was substitutedfor juvocimene. None of the compounds tested in FIG. 12A, 12B, or 12Cwere effective in the absence of ecdysone.

FXR Chimeras

A plasmid was constructed that expresses a hybrid protein (GGF)consisting of the human GR amino terminus and DBD linked to the carboxylterminus of FXR. GGF-dependent transcriptional activity was increased 3-and 15-fold by farnesol and CDCA (40 μM each), respectively (FIG. 12D).The limited GGF-dependent induction by farnesol was doubled bycotransfecting an RXR-expressing plasmid (FIG. 12D). Importantly, noactivity was detectable without GGF.

As shown in FIG. 12E, this FXR-like responsiveness led to tests ofnatural and synthetic insecticides as GGF effectors. In theseexperiments, CHO cells were transfected with receptor plasmidsexpressing GGF and mouse RXRα and a luciferase reporter plasmidcontaining the upstream 1.5 kbp of the MTV promoter. GGF-dependentactivity was stimulated between 2- and 7-fold in response to micromolardoses (e.g., 25 μM) of cypermethrin, diazinon, dieldrin, fipronil,piperonyl butoxide, rotenonic acid, cafestol, precocene, methyljasmonate, and abietic acid (FIG. 12E). Finally, esculetin inhibitedCDCA-induced GGF-dependent activity by 54% just as this precocene analogrepressed farnesol-induced activity in the assay using native FXR (datanot shown). These results indicate that the ligand binding domains ofFXR and EcR are required to mediate the transcriptional effects of JHsand insecticides.

While the invention has been described and illustrated with reference tocertain embodiments thereof, those skilled in the art will appreciatethat various changes, modifications and substitutions can be madetherein without departing from the spirit and scope of the invention.For example, effective dosages other than the dosages as set forthherein may be applicable as a consequence of variations in theresponsiveness insect population being treated. Likewise, the specificbiochemical responses observed may vary according to and depending onthe particular active compound selected or whether there are presentpharmaceutical carriers, as well as the type of formulation and mode ofadministration employed, and such expected variations or differences inthe results are contemplated in accordance with the objects andpractices of the present invention. All references referred to hereinare incorporated by reference in their entireties.

1. A method for testing the ability of a compound to act as a modulatorof insect growth comprising determining whether the compound increasesfarnesoid X-activated receptor (FXR)-mediated transcription and/orecdysone receptor (EcR)-mediated transcription.
 2. The method of claim1, wherein the increase of EcR-mediated transcription comprisespotentiation of hormone-activated EcR-mediated transcription.
 3. Themethod of claim 2, wherein the hormone comprises an ecdysteroid.
 4. Themethod of claim 3, wherein the hormone comprises muristerone A (murA) or20-hydroxyecdysone (20E).
 5. The method of claim 1, comprising the stepsof: (a) transfecting isolated cells with DNA comprising sequences thatencode a functional isoform of the ecdysone receptor (EcR) or thefarnesoid receptor (FXR); (b) cotransfecting the cell with: (i) DNAcomprising sequences that encode a functional heterologous bindingpartner for (a), wherein said heterologous binding partner complexeswith either EcR or FXR, and wherein said complex binds to a hormoneresponsive element (HRE) on DNA to activate gene transcription; and (ii)DNA comprising a reporter construct comprising a hormone responseelement (HRE) linked to a gene; (c) adding the compound to be tested;and (d) measuring an increase in the synthesis of the reporter geneprotein.
 6. The method of claim 5, wherein the increase in reporter geneprotein is used to quantify the ability of the test compound to increaseEcR-mediated transcription or FXR-mediated transcription.
 7. The methodof claim 6, wherein the ability of the test compound to activateEcR-mediated transcription or FXR-mediated transcription is correlatedto the potential insecticidal activity of the test compound.
 8. Themethod of claim 5, wherein the heterologous binding partner comprisesUltraspiracle (USP).
 9. The method of claim 5, wherein the heterologousbinding partner comprises a retinoid X receptor (RXR).
 10. The method ofclaim 5, further comprising adding a modulator of EcR activity to thecell.
 11. A method for in situ testing for the presence of compoundhaving the ability to act as a modulator of insect growth comprising:(a) transfecting a cell in an organism with DNA that encodes afunctional isoform of the farnesoid X-activated receptor (FXR) and/orthe ecdysone receptor (EcR); (b) cotransfecting the cell with: (i) DNAthat encodes a polypeptide comprising a functional retinoid X receptor(RXR) or Ultraspiracle (USP) isoform; and (ii) DNA comprising a hormoneresponse element (HRE) linked to a reporter gene; (c) optionally addingan ecdysteroid; (d) exposing the organism to a compound to be tested;and (e) measuring an increase in the protein encoded by the reportergene.
 12. The method of claim 11, wherein the organism comprises anatural or modified eukaryotic cell.
 13. The method of claim 11, whereinthe reporter construct comprises a promoter comprising multiple hormoneresponse elements (HREs) linked to a gene encoding a detectable geneproduct.
 14. The method of claim 11, wherein the reporter gene comprisesluciferase (LUC).
 15. The method of claim 11, wherein the hormoneresponse element comprises a EcR response element.
 16. The method ofclaim 11, wherein the hormone response element comprises a FXR responseelement.
 17. A composition for use as an insecticide comprising acompound that increases FXR-mediated transcription or EcR-mediatedtranscription mixed with a suitable carrier for application to plants.18. The composition of claim 17, wherein the increase in EcR-mediatedtranscription comprises potentiation of hormone-activated EcRtranscription.
 19. The composition of claim 17, wherein the hormonecomprises an ecdysteroid.